Methods and compositions for reducing nucleotide impurities

ABSTRACT

Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/108,179, filed Oct. 30, 2020, which is incorporated herein byreference in its entirety and for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 1, 2021, isnamed 051385-539001WO_SL_ST25.txt and is 11,825 bytes in size.

BACKGROUND

Modified nucleotides used in next generation sequencing (NGS)technologies, such as modified nucleotides containing reversibleterminators, often contain impurities, such as natural nucleotides ornon-reversible terminator-containing nucleotides. DNA polymerasesgenerally discriminate against modified nucleotides in favor of 3′-OHbearing nucleotide counterparts when presented as a mixture. Thistypically leads to the clusters of monoclonal amplicons beingout-of-phase, reducing sequencing accuracy and limiting sequencing readlengths.

BRIEF SUMMARY

In view of the foregoing, there is a need for an effective solution tothe synchrony problems in ensemble-based sequencing methods,particularly for long read lengths. Disclosed herein, inter alia, aresolutions to these and other problems in the art.

In an aspect is provided a composition including (a) nucleotidesincluding a free 3′-OH, (b) nucleotides lacking a free 3′-OH, and (c)one or more reagents for decreasing the amount of the nucleotidesincluding a free 3′-OH. In embodiments, the one or more reagents includea depletion primer, a depletion template, and a depletion polymerasethat is active to extend the depletion primer along the depletiontemplate by selectively incorporating the nucleotides including a free3′-OH, wherein the depletion primer and the depletion template are freein solution. In embodiments, the one or more reagents include one ormore nucleotide cyclases active to selectively cyclize the nucleotidesincluding a free 3′-OH.

In an aspect is provided a method of sequencing a target polynucleotide.In embodiments, the method includes (a) incubating the targetpolynucleotide in a composition described herein (e.g., a reactionmixture including a sequencing primer, nucleotides including a free3′-OH, nucleotides lacking a free 3′-OH, and a sequencing polymerase);(b) enzymatically decreasing the amount of the nucleotides including afree 3′-OH; (c) extending the sequencing primer along the targetpolynucleotide using the sequencing polymerase by incorporating one ofthe nucleotides lacking a free 3′-OH; and (d) identifying theincorporated nucleotide. In embodiments, steps (a)-(d) are performed ina sequencing flow cell. In embodiments, the target polynucleotide isimmobilized to a solid substrate.

In an aspect is provided a method of decreasing the amount of 3′-OHnucleotide in a sequencing solution, said method including: (a)contacting a sequencing solution with a depleting solution, wherein saidsequencing solution includes a 3′-OH nucleotide and a plurality oflabeled 3′-O-blocked reversible terminator nucleotides and wherein thedepleting solution includes: (i) a depletion polynucleotide and adepletion polymerase, wherein the depletion polymerase incorporates the3′-OH nucleotide into the depletion polynucleotide thereby producing anextended depletion polynucleotide; or (ii) a nucleotide cyclase, whereinthe nucleotide cyclase cyclizes the 3′-OH nucleotide thereby producing acyclized nucleotide; and (b) inactivating the depletion polymerase orthe nucleotide cyclase.

In an aspect is provided a method of increasing storage stability ofmodified nucleotides. In embodiments, the modified nucleotides are foruse in a sequencing reaction. In embodiments, the method of increasingthe storage stability includes (a) storing the modified nucleotides insolution at about 2° C.-65° C. for at least 12 hours, wherein themodified nucleotides include nucleotides lacking a free 3′-OH, andwherein the solution includes nucleotides including a free 3′-OH; and(b) depleting the nucleotides including a free 3′-OH during storage. Inembodiments, depleting the nucleotides including a free 3′-OH duringstorage includes extending a depletion primer along a depletion templateusing a depletion polymerase that selectively incorporates thenucleotides including a free 3′-OH, wherein the depletion primer and thedepletion template are free in solution. In embodiments, depleting thenucleotides including a free 3′-OH during storage includes selectivelycyclizing the nucleotides including the free 3′-OH using a nucleotidecyclase.

In an aspect is provided a method of sequencing a nucleic acid. Themethod includes (i) incorporating in series with a nucleic acidpolymerase, within a reaction vessel, one of four different nucleotidesfrom a composition (e.g., a nucleotide solution) described herein into aprimer to create an extension strand, wherein said primer is hybridizedto the nucleic acid and wherein each of the four different nucleotidescomprises a detectable moiety; and (ii) detecting the detectable moietyof each incorporated nucleotide, so as to thereby identify eachincorporated nucleotide in said extension strand, thereby sequencing thenucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . A simplified depiction of a desired sequencing-by-synthesis(SBS) in-phase nucleotide incorporation and detection event. Thecomplementary strand is extended by adding a modified G nucleotide,wherein the G nucleotide contains a 3′ reversible terminator (RT) and alabel (pictorially represented as a spiky ball). Note, the modified Gnucleotide is incorporated via a polymerase, though the polymerase isnot shown in the illustration. Following detection, the RT and linkerare cleaved thereby removing the label and exposing a free 3′ hydroxylgroup.

FIG. 2 . A simplified depiction of an undesired out-of-phase nucleotideincorporation and detection event. The complementary strand is extendedby adding a modified G nucleotide, wherein the G nucleotide contains alabel, but is unterminated (i.e., does not contain a 3′-RT, rather afree 3′-OH). An additional modified nucleotide present in solution(shown as a modified C in the illustration) may then extend thecomplementary strand further. During the detection event, the labels forboth the first incorporated nucleotide (G) and the second incorporatednucleotide (C) are detected; both labels are then removed, optionallysimultaneously along with the RT. The resulting complementary strand isout of phase with the surrounding amplicons.

FIGS. 3A-3B. Depicted in FIG. 3A is an embodiment of removingnon-terminated (i.e., nucleotides without 3′-RTs) from a nucleotidesolution. The nucleotide solution includes labeled nucleotidescontaining a 3′-RT moiety, and nucleotides containing 3′-hydroxylmoieties. In the presence of a depletion template and a depletionpolymerase not capable of incorporating modified nucleotides (e.g.,Klenow), the non-terminated nucleotides are incorporated into thedepletion template (not shown). The resulting depleted nucleotidesolution no longer contains non-terminated nucleotides (as depicted inFIG. 3B).

FIGS. 4A-4B. Effects of nucleotide depletion (also referred to aslive-polishing) on percent lead. FIG. 4A shows the results ofreversibly-terminated nucleotides stored at either 4° C. or 37° C. for1, 3, or 7 days (1 week) assayed for non-terminated nucleotideincorporation in the absence of a depletion solution (i.e., a non-livepolished solution). Freshly manufactured, (F) nucleotides were includedas a no-storage, non-depleted control. FIG. 4B shows the results ofreversibly-terminated nucleotides stored at either 4° C. or 37° C. for1, 3, or 7 days (1 week) assayed for non-terminated nucleotideincorporation in the presence of a depletion solution.

FIG. 5 . The quality score plotted per cycle of nucleotide incorporationfrom a 50-cycle sequencing run comparing no Klenow enzyme to a 3× and10× concentration of Klenow enzyme.

DETAILED DESCRIPTION

The aspects and embodiments described herein relate to depletingnucleotide impurities from nucleotide solutions. For example, describedherein is a nucleotide solution. In embodiments, the nucleotide solutionincludes a plurality of modified nucleotides, depletion enzyme, buffer,and salt. In embodiments, the modified nucleotides include labeledreversibly-terminated nucleotides and labeled nucleotides. Inembodiments, the solution includes a depletion oligonucleotide templateand the depletion enzyme is a polymerase. In embodiments, the depletionenzyme is a cyclase. In embodiments, the nucleotide solution ismaintained at a temperature of 4° C., 25° C., or 65° C. In embodiments,the depletion enzyme is active at low temperatures (e.g., 2° C.-10° C.).In embodiments, the depletion enzyme is not thermostable. Inembodiments, the depletion enzyme is a zinc metalloenzyme. Inembodiments, the depletion oligonucleotide templates are hairpins with a5′-overhang with a poly(N) sequence, where N is T, G, C, or A. Asdescribed herein, the term “depletion oligonucleotide template” may beused interchangeably with “depletion template”.

Described herein is a method of decreasing the amount of non-terminatedmodified nucleotides from a stored nucleotide solution. A storednucleotide solution is a nucleotide solution that has been stored for aperiod of time (e.g., at least one day) following nucleotidemanufacturing. In a stored nucleotide solution, the percentage ofnon-terminated nucleotides may increase relative to a freshlymanufactured and purified nucleotide solution. In embodiments, thestored nucleotide solution is maintained at 2-8° C. or 20-30° C. Inembodiments, the method includes mixing a stored nucleotide solutioncontaining non-terminated and reversibly-terminated nucleotides with adepletion enzyme, wherein the depletion enzyme catalyzes a reaction withthe non-terminated modified nucleotides to decrease the amount ofnon-terminated nucleotides from the stored nucleotide solution.

Also described herein is a method of decreasing the amount ofnon-terminated modified nucleotides from a sequencing solution within amicrofluidic device. In embodiments, the method includes mixing asequencing solution containing non-terminated and reversibly-terminatednucleotides with a depletion enzyme, wherein the depletion enzymecatalyzes a reaction with the non-terminated modified nucleotides todecrease the amount of non-terminated nucleotides from the sequencingsolution; and flowing the sequencing solution into a reaction vesselwithin the microfluidic device.

I. Definitions

The practice of the technology described herein will employ, unlessindicated specifically to the contrary, conventional methods ofchemistry, biochemistry, organic chemistry, molecular biology,microbiology, recombinant DNA techniques, genetics, immunology, and cellbiology that are within the skill of the art, many of which aredescribed below for the purpose of illustration. Examples of suchtechniques are available in the literature. Methods, devices andmaterials similar or equivalent to those described herein can be used inthe practice of this invention.

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey affect the activity or action of the listed elements.

As used herein, the term “control” or “control experiment” is used inaccordance with its plain and ordinary meaning and refers to anexperiment in which the subjects or reagents of the experiment aretreated as in a parallel experiment except for omission of a procedure,reagent, or variable of the experiment. In some instances, the controlis used as a standard of comparison in evaluating experimental effects.For example the methods described herein measure an increase or decreaseof a property relative to a control and it is understood that one havingskill in the art would conduct a parallel experiment while omitting oneor more steps of the method (e.g., omitting contact with one or moredepleting reagents).

As used herein, the term “complement” is used in accordance with itsplain and ordinary meaning and refers to a nucleotide (e.g., RNAnucleotide or DNA nucleotide) or a sequence of nucleotides capable ofbase pairing with another nucleotide or sequence of nucleotides (e.g.,Watson-Crick base pairing). As described herein and commonly known inthe art the complementary (matching) nucleotide of adenosine isthymidine in DNA, or alternatively in RNA the complementary (matching)nucleotide of adenosine is uracil, and the complementary (matching)nucleotide of guanosine is cytosine. Thus, a complement may include asequence of nucleotides that base pair with corresponding complementarynucleotides of a second nucleic acid sequence. The nucleotides of acomplement may partially or completely match the nucleotides of thesecond nucleic acid sequence. Where the nucleotides of the complementcompletely match each nucleotide of the second nucleic acid sequence,the complement forms base pairs with each nucleotide of the secondnucleic acid sequence. Where the nucleotides of the complement partiallymatch the nucleotides of the second nucleic acid sequence only some ofthe nucleotides of the complement form base pairs with nucleotides ofthe second nucleic acid sequence. Examples of complementary sequencesinclude coding and non-coding sequences, wherein the non-coding sequencecontains complementary nucleotides to the coding sequence and thus formsthe complement of the coding sequence. A further example ofcomplementary sequences are sense and antisense sequences, wherein thesense sequence contains complementary nucleotides to the antisensesequence and thus forms the complement of the antisense sequence.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,sequences in a pair of complementary sequences form portions of a singlepolynucleotide with non-base-pairing nucleotides (e.g., as in a hairpinstructure, with or without an overhang) or portions of separatepolynucleotides. In embodiments, one or both sequences in a pair ofcomplementary sequences form portions of longer polynucleotides, whichmay or may not include additional regions of complementarity.

As used herein, the term “contacting” is used in accordance with itsplain ordinary meaning and refers to the process of allowing at leasttwo distinct species (e.g. chemical compounds including biomolecules orcells) to become sufficiently proximal to react, interact or physicallytouch. However, the resulting reaction product can be produced directlyfrom a reaction between the added reagents or from an intermediate fromone or more of the added reagents that can be produced in the reactionmixture. The term “contacting” may include allowing two species toreact, interact, or physically touch, wherein the two species may be acompound, nucleic acid, a protein, or enzyme (e.g., a DNA polymerase).

As used herein, the term “nucleic acid” is used in accordance with itsplain and ordinary meaning and refers to nucleotides (e.g.,deoxyribonucleotides or ribonucleotides) and polymers thereof in eithersingle-, double- or multiple-stranded form, or complements thereof. Theterms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, inthe usual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences. A“nucleoside” is structurally similar to a nucleotide, but is missing thephosphate moieties. An example of a nucleoside analogue would be one inwhich the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

As used herein, the term “modified nucleotide” refers to nucleotidemodified in some manner. Typically, a nucleotide contains a single5-carbon sugar moiety, a single nitrogenous base moiety and 1 to threephosphate moieties. In embodiments, a nucleotide can include a blockingmoiety or a label moiety. A blocking moiety on a nucleotide preventsformation of a covalent bond between the 3′ hydroxyl moiety of thenucleotide and the 5′ phosphate of another nucleotide. A blocking moietyon a nucleotide can be reversible, whereby the blocking moiety can beremoved or modified to allow the 3′ hydroxyl to form a covalent bondwith the 5′ phosphate of another nucleotide. A blocking moiety can beeffectively irreversible under particular conditions used in a methodset forth herein. A label moiety of a nucleotide can be any moiety thatallows the nucleotide to be detected, for example, using a spectroscopicmethod. Exemplary label moieties are fluorescent labels, mass labels,chemiluminescent labels, electrochemical labels, detectable labels andthe like. One or more of the above moieties can be absent from anucleotide used in the methods and compositions set forth herein. Forexample, a nucleotide can lack a label moiety or a blocking moiety orboth. Examples of nucleotide analogues include, without limitation,7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotidesshown herein, analogues in which a label is attached through a cleavablelinker to the 5-position of cytosine or thymine or to the 7-position ofdeaza-adenine or deaza-guanine, and analogues in which a small chemicalmoiety is used to cap the —OH group at the 3′-position of deoxyribose.Nucleotide analogues and DNA polymerase-based DNA sequencing are alsodescribed in U.S. Pat. No. 6,664,079, which is incorporated herein byreference in its entirety for all purposes

The term “primer,” as used herein, refers to an oligonucleotide, eithernatural or synthetic, which is capable, upon forming a duplex with apolynucleotide (e.g., the target polynucleotide or the depletiontemplate), of acting as a point of initiation of nucleic acid synthesisand being extended from one of its ends along the template so that anextended polynucleotide duplex is formed. The sequence of nucleotidesadded during the extension process is determined by the sequence of thepolynucleotide. Primers usually are extended by a DNA polymerase. Aprimer can be of any length depending on the particular technique itwill be used for. For example, PCR primers are generally between 10 and40 nucleotides in length. The length and complexity of the nucleic acidfixed onto the nucleic acid template is not critical to the invention.One of skill can adjust these factors to provide optimum hybridizationand signal production for a given hybridization procedure, and toprovide the required resolution among different genes or genomiclocations. The primer permits the addition of a nucleotide residuethereto, or oligonucleotide or polynucleotide synthesis therefrom, undersuitable conditions known in the art. In an embodiment the primer is aDNA primer, i.e., a primer consisting of, or largely consisting of,deoxyribonucleotide residues. The primers are designed to have asequence that is the complement of a region of DNA (e.g., the depletiontemplate or the target polynucleotide) to which the primer hybridizes.The addition of a nucleotide residue to the 3′ end of a primer byformation of a phosphodiester bond results in a DNA extension product.The addition of a nucleotide residue to the 3′ end of the DNA extensionproduct by formation of a phosphodiester bond results in a further DNAextension product. In another embodiment the primer is an RNA primer.

Nucleic acids, including e.g., nucleic acids with a phosphothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent, or otherinteraction.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

As used herein, the terms “analogue” and “analog”, in reference to achemical compound, refers to compound having a structure similar to thatof another one, but differing from it in respect of one or moredifferent atoms, functional groups, or substructures that are replacedwith one or more other atoms, functional groups, or substructures. Inthe context of a nucleotide, a “nucleotide analog” and “modifiednucleotide” refer to a compound that, like the nucleotide of which it isan analog, can be incorporated into a nucleic acid molecule (e.g., anextension product) by a suitable polymerase, for example, a DNApolymerase in the context of a nucleotide analogue. The terms alsoencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,or non-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, include,without limitation, phosphodiester derivatives including, e.g.,phosphoramidate, phosphorodiamidate, phosphorothioate (also known asphosphothioate having double bonded sulfur replacing oxygen in thephosphate), phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see, e.g., see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: APRACTICAL APPROACH, Oxford University Press) as well as modifications tothe nucleotide bases such as in 5-methyl cytidine or pseudouridine; andpeptide nucleic acid backbones and linkages. Other analog nucleic acidsinclude those with positive backbones; non-ionic backbones, modifiedsugars, and non-ribose backbones (e.g. phosphorodiamidate morpholinooligos or locked nucleic acids (LNA)), including those described in U.S.Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC SymposiumSeries 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui &Cook, eds. Nucleic acids containing one or more carbocyclic sugars arealso included within one definition of nucleic acids. Modifications ofthe ribose-phosphate backbone may be done for a variety of reasons,e.g., to increase the stability and half-life of such molecules inphysiological environments or as probes on a biochip. Mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Inembodiments, the intemucleotide linkages in DNA are phosphodiester,phosphodiester derivatives, or a combination of both.

As used herein, a “native” nucleotide is used in accordance with itsplain and ordinary meaning and refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as those that maycharacterize a nucleotide analog (e.g., an analog having a reversibleterminating moiety).

In embodiments, the nucleotides of the present disclosure use acleavable linker to attach the label to the nucleotide. The use of acleavable linker ensures that the label can, if required, be removedafter detection, avoiding any interfering signal with any labellednucleotide incorporated subsequently. The use of the term “cleavablelinker” is not meant to imply that the whole linker is required to beremoved from the nucleotide base. The cleavage site can be located at aposition on the linker that ensures that part of the linker remainsattached to the nucleotide base after cleavage. The linker can beattached at any position on the nucleotide base provided thatWatson-Crick base pairing can still be carried out. In the context ofpurine bases, it is preferred if the linker is attached via the7-position of the purine or the preferred deazapurine analogue, via an8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5-position oncytidine, thymidine or uracil and the N-4 position on cytosine. The term“cleavable linker” or “cleavable moiety” as used herein refers to adivalent or monovalent, respectively, moiety which is capable of beingseparated (e.g., detached, split, disconnected, hydrolyzed, a stablebond within the moiety is broken) into distinct entities. A cleavablelinker is cleavable (e.g., specifically cleavable) in response toexternal stimuli (e.g., enzymes, nucleophilic/basic reagents, reducingagents, photo-irradiation, electrophilic/acidic reagents, organometallicand metal reagents, or oxidizing reagents). A chemically cleavablelinker refers to a linker which is capable of being split in response tothe presence of a chemical (e.g., acid, base, oxidizing agent, reducingagent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid,fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄),or hydrazine (N₂H₄)). A chemically cleavable linker is non-enzymaticallycleavable. In embodiments, the cleavable linker is cleaved by contactingthe cleavable linker with a cleaving agent. In embodiments, the cleavingagent is a phosphine containing reagent (e.g., TCEP or THPP), sodiumdithionite (Na₂S₂O₄), weak acid, hydrazine (N₂H₄), Pd(0), orlight-irradiation (e.g., ultraviolet radiation).

As used herein, the terms “blocking moiety,” “reversible blockinggroup,” “reversible terminator” and “reversible terminator moiety” areused in accordance with their plain and ordinary meanings and refer to acleavable moiety which does not interfere with incorporation of anucleotide comprising it by a polymerase (e.g., DNA polymerase, modifiedDNA polymerase), but prevents further strand extension until removed(“unblocked”). For example, a reversible terminator may refer to ablocking moiety located, for example, at the 3′ position of thenucleotide and may be a chemically cleavable moiety such as an allylgroup, an azidomethyl group or a methoxymethyl group, or may be anenzymatically cleavable group such as a phosphate ester. Suitablenucleotide blocking moieties are described in applications WO2004/018497, U.S. Pat. Nos. 7,057,026, 7,541,444, WO 96/07669, U.S. Pat.Nos. 5,763,594, 5,808,045, 5,872,244 and 6,232,465 the contents of whichare incorporated herein by reference in their entirety. The nucleotidesmay be labelled or unlabelled. The nucleotides may be modified withreversible terminators useful in methods provided herein and may be3′-O-blocked reversible or 3′-unblocked reversible terminators. Innucleotides with 3′-O-blocked reversible terminators, the blocking groupmay be represented as —OR [reversible terminating (capping) group],wherein 0 is the oxygen atom of the 3′-OH of the pentose and R is theblocking group, while the label is linked to the base, which acts as areporter and can be cleaved. The 3′-O-blocked reversible terminators areknown in the art, and may be, for instance, a 3′-ONH₂ reversibleterminator, a 3′-O-allyl reversible terminator, or a 3′-O-azidomethylreversible terminator. In embodiments the reversible terminator moietyis

In embodiments, the reversible terminator moiety is

as described in U.S. Pat. No. 10,738,072, which is incorporated hereinby reference for all purposes.

As used herein, the term “label” or “labels” is used in accordance withtheir plain and ordinary meanings and refer to molecules that candirectly or indirectly produce or result in a detectable signal eitherby themselves or upon interaction with another molecule. Non-limitingexamples of detectable labels include fluorescent dyes, biotin, digoxin,haptens, and epitopes. In general, a dye is a molecule, compound, orsubstance that can provide an optically detectable signal, such as acolorimetric, luminescent, bioluminescent, chemiluminescent,phosphorescent, or fluorescent signal. In embodiments, the label is adye. In embodiments, the dye is a fluorescent dye. Non-limiting examplesof dyes, some of which are commercially available, include CF dyes(Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (ThermoFisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.),and HiLyte dyes (Anaspec, Inc.). In embodiments, a particular nucleotidetype is associated with a particular label, such that identifying thelabel identifies the nucleotide with which it is associated. Inembodiments, the label is luciferin that reacts with luciferase toproduce a detectable signal in response to one or more bases beingincorporated into an elongated complementary strand, such as inpyrosequencing. In embodiment, a nucleotide comprises a label (such as adye). In embodiments, the label is not associated with any particularnucleotide, but detection of the label identifies whether one or morenucleotides having a known identity were added during an extension step(such as in the case of pyrosequencing).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics,which are not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may In embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein, which encodes apolypeptide, also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid that encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosure.

The following groups each contain amino acids that are conservativesubstitutions for one another: 1) Non-polar—Alanine (A), Leucine (L),Isoleucine (I), Valine (V), Glycine (G), Methionine (M); 2)Aliphatic—Alanine (A), Leucine (L), Isoleucine (I), Valine (V); 3)Acidic—Aspartic acid (D), Glutamic acid (E); 4) Polar—Asparagine (N),Glutamine (Q); Serine (S), Threonine (T); 5) Basic—Arginine (R), Lysine(K); 7) Aromatic—Phenylalanine (F), Tyrosine (Y), Tryptophan (W),Histidine (H); 8) Other—Cystein (C) and Proline (P).

The term “amino acid side chain” refers to the functional substituentcontained on amino acids. For example, an amino acid side chain may bethe side chain of a naturally occurring amino acid. Naturally occurringamino acids are those encoded by the genetic code (e.g., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, orvaline), as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In embodiments,the amino acid side chain may be a non-natural amino acid side chain.

The term “non-natural amino acid side chain” refers to the functionalsubstituent of compounds that have the same basic chemical structure asa naturally occurring amino acid, i.e., an a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, allylalanine, 2-aminoisobutryric acid. Non-natural aminoacids are non-proteinogenic amino acids that occur naturally or arechemically synthesized. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Non-limitingexamples include exo-cis-3-Aminobicyclo[2.2.1]hept-5-ene-2-carboxylicacid hydrochloride, cis-2-Aminocycloheptanecarboxylic acidhydrochloride,cis-6-Amino-3-cyclohexene-1-carboxylic acid hydrochloride,cis-2-Amino-2-methylcyclohexanecarboxylic acid hydrochloride,cis-2-Amino-2-methylcyclopentanecarboxylic acid hydrochloride,2-(Boc-aminomethyl)benzoic acid, 2-(Boc-amino)octanedioic acid,Boc-4,5-dehydro-Leu-OH (dicyclohexylammonium),Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-β-Homopyr-OH,Boc-(2-indanyl)-Gly-OH, 4-Boc-3-morpholineacetic acid,4-Boc-3-morpholineacetic acid, Boc-pentafluoro-D-phenylalanine,Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)—OH, Boc-Phe(4-Br)—OH,Boc-D-Phe(4-Br)—OH, Boc-D-Phe(3-Cl)—OH, Boc-Phe(4-NH2)-OH,Boc-Phe(3-NO2)-OH, Boc-Phe(3,5-F2)-OH,2-(4-Boc-piperazino)-2-(3,4-dimethoxyphenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(3-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid purum,2-(4-Boc-piperazino)-2-(4-methoxyphenyl)acetic acid purum,2-(4-Boc-piperazino)-2-phenylacetic acid purum,2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid purum,2-(4-Boc-piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid purum,Boc-β-(2-quinolyl)-Ala-OH, N-Boc-1,2,3,6-tetrahydro-2-pyridinecarboxylicacid, Boc-β-(4-thiazolyl)-Ala-OH, Boc-β-(2-thienyl)-D-Ala-OH,Fmoc-N-(4-Boc-aminobutyl)-Gly-OH, Fmoc-N-(2-Boc-aminoethyl)-Gly-OH,Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, Fmoc-(2-indanyl)-Gly-OH,Fmoc-pentafluoro-L-phenylalanine, Fmoc-Pen(Trt)-OH, Fmoc-Phe(2-Br)—OH,Fmoc-Phe(4-Br)—OH, Fmoc-Phe(3,5-F2)-OH, Fmoc-β-(4-thiazolyl)-Ala-OH,Fmoc-β-(2-thienyl)-Ala-OH, 4-(Hydroxymethyl)-D-phenylalanine.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see.e.g., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. As used herein, percent (%) aminoacid sequence identity is defined as the percentage of amino acids in acandidate sequence that are identical to the amino acids in a referencesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 700, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

The terms “position”, “numbered with reference to” or “correspondingto,” when used in the context of the numbering of a given amino acid orpolynucleotide sequence, refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. As used herein, the term“functionally equivalent to” in relation to an amino acid positionrefers to an amino acid residue in a protein that corresponds to aparticular amino acid in a reference sequence. An amino acid“corresponds” to a given residue when it occupies the same essentialstructural position within the protein as the given residue. One skilledin the art will immediately recognize the identity and location ofresidues corresponding to a specific position in a protein (e.g.,polymerase) in other proteins with different numbering systems. Forexample, by performing a simple sequence alignment with a protein (e.g.,polymerase) the identity and location of residues corresponding tospecific positions of said protein are identified in other proteinsequences aligning to said protein. Due to deletions, insertions,truncations, fusions, and the like that must be taken into account whendetermining an optimal alignment, in general the amino acid residuenumber in a test sequence determined by simply counting from theN-terminus will not necessarily be the same as the number of itscorresponding position in the reference sequence. For example, in a casewhere a variant has a deletion relative to an aligned referencesequence, there will be no amino acid in the variant that corresponds toa position in the reference sequence at the site of deletion. Wherethere is an insertion in an aligned reference sequence, that insertionwill not correspond to a numbered amino acid position in the referencesequence. In the case of truncations or fusions there can be stretchesof amino acids in either the reference or aligned sequence that do notcorrespond to any amino acid in the corresponding sequence.

“Polymerase,” as used herein, refers to any natural or non-naturallyoccurring enzyme or other catalyst that is capable of catalyzing apolymerization reaction, such as the polymerization of nucleotidemonomers to form a nucleic acid polymer. Exemplary types of polymerasesthat may be used in the compositions and methods of the presentdisclosure include the nucleic acid polymerases such as DNA polymerase,DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In somecases, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0DNA polymerase, 9° N polymerase (exo-)A485L/Y409V, Phi29 DNA Polymerase(φ29 DNA Polymerase), T7 DNA polymerase, DNA polymerase II, DNApolymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentRDNA polymerase, Therminator™ II DNA Polymerase, Therminator™ III DNAPolymerase, or Therminator™ IX DNA Polymerase. In embodiments, thepolymerase is a protein polymerase. Typically, a DNA polymerase addsnucleotides to the 3′-end of a DNA strand, one nucleotide at a time. Inembodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNApolymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNApolymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNApolymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNApolymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNApolymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNApolymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or athermophilic nucleic acid polymerase (e.g. Therminator γ, 9° Npolymerase (exo-), Therminator II, Therminator III, or Therminator IX).As used herein, the term “thermophilic nucleic acid polymerase” refersto a family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase including its high strand displacement activity. The sequenceAIA (D141A, E143A) was chosen for reducing exonuclease. Subsequentmutagenesis of key amino acids results in an increased ability of theenzyme to incorporate dideoxynucleotides, ribonucleotides andacyclonucleotides (e.g., Therminator II enzyme from New England Biolabswith D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs, 3′-azido-dNTPsand other 3′-modified nucleotides (e.g., NEB Therminator III DNAPolymerase with D141A/E143A/L408S/Y409A/P410V mutations, NEB TherminatorIX DNA polymerase), or γ-phosphate labeled nucleotides (e.g.,Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.ChemBioChem. 2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J, et al.Proceedings of the National Academy of Sciences of the United States ofAmerica. 2008; 105(27):9145-9150), which are incorporated herein intheir entirety for all purposes.

As used herein, the term “exonuclease activity” is used in accordancewith its ordinary meaning in the art, and refers to the removal of anucleotide from a nucleic acid by a DNA polymerase. For example, duringpolymerization, nucleotides are added to the 3′ end of the primerstrand. Occasionally a DNA polymerase incorporates an incorrectnucleotide to the 3′-OH terminus of the primer strand, wherein theincorrect nucleotide cannot form a hydrogen bond to the correspondingbase in the template strand. Such a nucleotide, added in error, isremoved from the primer as a result of the 3′ to 5′ exonuclease activityof the DNA polymerase. In embodiments, exonuclease activity may bereferred to as “proofreading.” When referring to 3′-5′ exonucleaseactivity, it is understood that the DNA polymerase facilitates ahydrolyzing reaction that breaks phosphodiester bonds at either the 3′end of a polynucleotide chain to excise the nucleotide. In embodiments,3′-5′ exonuclease activity refers to the successive removal ofnucleotides in single-stranded DNA in a 3′→5′ direction, releasingdeoxyribonucleoside 5′-monophosphates one after another. Methods forquantifying exonuclease activity are known in the art, see for exampleSouthworth et al, PNAS Vol 93, 8281-8285 (1996).

As used herein, the term “incorporating” or “chemically incorporating,”when used in reference to a primer and cognate nucleotide, refers to theprocess of joining the cognate nucleotide to the primer or extensionproduct thereof by formation of a phosphodiester bond.

As used herein, the term “selective” or “selectivity” or the like of acompound refers to the compound's ability to discriminate betweenmolecular targets. For example, in a pool of nucleotides in which somenucleotides have a free 3′-OH and other nucleotides do not, an enzymethat selectively acts upon the nucleotides having a free 3′-OH is lesslikely to act (or not capable of acting) upon the nucleotides lacking afree 3′-OH. As a result, following the action by the enzyme, therelative proportion of nucleotides having a free 3′-OH in the pool isdecreased. In embodiments, the enzyme is a polymerase that selectivelyincorporates nucleotides having a free 3′-OH in a primer extensionreaction. In embodiments, the enzyme is a nucleotide cyclase thatselectively cyclizes the nucleotides having a free 3′-OH.

As used herein, the terms “specific”, “specifically”, “specificity”, orthe like of a compound refers to the compound's ability to cause aparticular action, such as binding, to a particular molecular targetwith minimal or no action to other proteins in the cell.

As used herein, the terms “bind” and “bound” are used in accordance withtheir plain and ordinary meanings and refer to an association betweenatoms or molecules. The association can be direct or indirect. Forexample, bound atoms or molecules may be directly bound to one another,e.g., by a covalent bond or non-covalent bond (e.g. electrostaticinteractions (e.g. ionic bond, hydrogen bond, halogen bond), van derWaals interactions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). As a further example, two molecules may be bound indirectlyto one another by way of direct binding to one or more intermediatemolecules, thereby forming a complex.

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof partial as well as full sequence information, including theidentification, ordering, or locations of the nucleotides that comprisethe polynucleotide being sequenced, and inclusive of the physicalprocesses for generating such sequence information. That is, the termincludes sequence comparisons, fingerprinting, and like levels ofinformation about a target polynucleotide, as well as the expressidentification and ordering of nucleotides in a target polynucleotide.The term also includes the determination of the identification,ordering, and locations of one, two, or three of the four types ofnucleotides within a target polynucleotide. Sequencing methods, such asthose outlined in U.S. Pat. No. 5,302,509 can be carried out using thenucleotides described herein. The sequencing methods are preferablycarried out with the target polynucleotide arrayed on a solid substrate.Multiple target polynucleotides can be immobilized on the solid supportthrough linker molecules, or can be attached to particles, e.g.,microspheres, which can also be attached to a solid substrate. Inembodiments, the solid substrate is in the form of a chip, a bead, awell, a capillary tube, a slide, a wafer, a filter, a fiber, a porousmedia, or a column. In embodiments, the solid substrate is gold, quartz,silica, plastic, glass, diamond, silver, metal, or polypropylene. Inembodiments, the solid substrate is porous.

As used herein, the term “sequencing reaction mixture” or “sequencingsolution” is used in accordance with its plain and ordinary meaning andrefers to an aqueous mixture that contains the reagents necessary toallow a dNTP or dNTP analogue to add a nucleotide to a DNA strand by aDNA polymerase. In embodiments, the sequencing reaction mixture includesa buffer. In embodiments, the buffer includes an acetate buffer,3-(N-morpholino)propanesulfonic acid (MOPS) buffer,N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer. In embodiments, the sequencing reaction mixture includesnucleotides, wherein the nucleotides include a reversible terminatingmoiety and a label covalently linked to the nucleotide via a cleavablelinker. In embodiments, the sequencing reaction mixture includes abuffer, DNA polymerase, detergent (e.g., Triton X), a chelator (e.g.,EDTA), and/or salts (e.g., ammonium sulfate, magnesium chloride, sodiumchloride, or potassium chloride). In embodiments, the sequencingreaction mixture includes the reagents used in a sequencing-by-synthesisprotocol.

As used herein, the term “sequencing cycle” is used in accordance withits plain and ordinary meaning and refers to incorporating one or morenucleotides (e.g., nucleotide analogues) to the 3′ end of apolynucleotide with a polymerase, and detecting one or more labels thatidentify the one or more nucleotides incorporated. The sequencing may beaccomplished by, for example, sequencing by synthesis, pyrosequencing,and the like. In embodiments, a sequencing cycle includes extending acomplementary polynucleotide by incorporating a first nucleotide using apolymerase, wherein the polynucleotide is hybridized to a templatenucleic acid, detecting the first nucleotide, and identifying the firstnucleotide. In embodiments, to begin a sequencing cycle, one or moredifferently labeled nucleotides and a DNA polymerase can be introduced.Following nucleotide addition, signals produced (e.g., via excitationand emission of a detectable label) can be detected to determine theidentity of the incorporated nucleotide (based on the labels on thenucleotides). Reagents can then be added to remove the 3′ reversibleterminator and to remove labels from each incorporated base. Reagents,enzymes and other substances can be removed between steps by washing.Cycles may include repeating these steps, and the sequence of eachcluster is read over the multiple repetitions.

“Hybridize” shall mean the annealing of a nucleic acid sequence toanother nucleic acid sequence (e.g., one single-stranded nucleic acid(such as a primer) to another nucleic acid) based on the well-understoodprinciple of sequence complementarity. In an embodiment the othernucleic acid is a single-stranded nucleic acid. In some embodiments, oneportion of a nucleic acid hybridizes to itself, such as in the formationof a hairpin structure. The propensity for hybridization between nucleicacids depends on the temperature and ionic strength of their milieu, thelength of the nucleic acids and the degree of complementarity. Theeffect of these parameters on hybridization is described in, forexample, Sambrook J., Fritsch E. F., Maniatis T., Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York (1989).As used herein, hybridization of a primer, or of a DNA extensionproduct, respectively, is extendable by creation of a phosphodiesterbond with an available nucleotide or nucleotide analogue capable offorming a phosphodiester bond, therewith. For example, hybridization canbe performed at a temperature ranging from 15° C. to 95° C. In someembodiments, the hybridization is performed at a temperature of about20° C., about 25° C., about 30° C., about 35° C., about 40° C., about45° C., about 50° C., about 55° C., about 60° C., about 65° C., about70° C., about 75° C., about 80° C., about 85° C., about 90° C., or about95° C. In other embodiments, the stringency of the hybridization can befurther altered by the addition or removal of components of the bufferedsolution.

As used herein, the term “extension” or “elongation” is used inaccordance with their plain and ordinary meanings and refer to synthesisby a polymerase of a new polynucleotide strand complementary to atemplate strand by adding free nucleotides (e.g., dNTPs) from a reactionmixture that are complementary to the template in the 5′-to-3′direction. Extension includes condensing the 5′-phosphate group of thedNTPs with the 3′-hydroxy group at the end of the nascent (elongating)DNA strand.

The term “reaction vessel” is used in accordance with its ordinarymeaning in chemistry or chemical engineering, and refers to a containerhaving an inner volume in which a reaction takes place. In embodiments,the reaction vessel may be designed to provide suitable reactionconditions such as reaction volume, reaction temperature or pressure,and stirring or agitation, which may be adjusted to ensure that thereaction proceeds with a desired, sufficient or highest efficiency forproducing a product from the chemical reaction. In embodiments, thereaction vessel is a container for liquid, gas or solid. In embodiments,the reaction vessel may include an inlet, an outlet, a reservoir and thelike. In embodiments, the reaction vessel is connected to a pump (e.g.,vacuum pump), a controller (e.g., CPU), or a monitoring device (e.g., UVdetector or spectrophotometer).

As used herein, the term “sequencing read” is used in accordance withits plain and ordinary meaning and refers to an inferred sequence ofbase pairs (or base pair probabilities) corresponding to all or part ofa single DNA fragment. Sequencing technologies vary in the length ofreads produced. Reads of length 20-40 base pairs (bp) are referred to asultra-short. Typical sequencers produce read lengths in the range of100-500 bp. A sequencing read may include 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, or more nucleotide bases. Read length is afactor which can affect the results of biological studies. For example,longer read lengths improve the resolution of de novo genome assemblyand detection of structural variants.

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid or an agent may be affixed. Non-limitingexamples include chips, beads and columns. The solid substrate can benon-porous or porous. Exemplary solid substrates include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers. In embodiments, the solid substrate has at least one surfacelocated within a flow cell. The solid substrate, or regions thereof, canbe substantially flat. The solid substrate can have surface featuressuch as wells, pits, channels, ridges, raised regions, pegs, posts orthe like. The term solid substrate is encompassing of a substrate (e.g.,a flow cell) having a surface comprising a polymer coating covalentlyattached thereto. In embodiments, the solid substrate is a flow cell.The term “flow cell” as used herein refers to a chamber including asolid surface across which one or more fluid reagents can be flowed.Examples of flow cells and related fluidic systems and detectionplatforms that can be readily used in the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008). The term “flow cell” may refer to the reaction vesselin a microfluidic device (e.g., nucleic acid sequencing device). Theflow cell is typically a glass slide containing small fluidic channels(e.g., a glass slide 75 mm×25 mm×1 mm having one or more channels),through which sequencing solutions (e.g., polymerases, nucleotides, andbuffers) may traverse. Though typically glass, suitable flow cellmaterials may include polymeric materials, plastics, silicon, quartz(fused silica), Borofloat® glass, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, sapphire, orplastic materials such as COCs and epoxies. The particular material canbe selected based on properties desired for a particular use. The flowcells used in the various embodiments can include millions of individualnucleic acid clusters, e.g., about 2-8 million clusters per channel.Each of such clusters can give read lengths of at least 25-100 bases forDNA sequencing. The systems and methods herein can generate over agigabase (one billion bases) of sequence per run.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The methods and kits of the present disclosure may be applied, mutatismutandis, to the sequencing of RNA, or to determining the identity of aribonucleotide.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g.,packaging, buffers, written instructions for performing a method, etc.)from one location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to a delivery system comprising two or more separate containersthat each contain a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

As used herein, the terms “reduce,” “decrease,” “reduction,” “minimal,”“low,” or “lower” refer to decreases below basal levels, e.g., ascompared to a control. The terms “increase,” high,” “higher,” “maximal,”“elevate,” or “elevation” refer to increases above basal levels, e.g.,as compared to a control. Increases, elevations, decreases, orreductions can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% compared to a control or standard level. Each ofthe values or ranges recited herein may include any value or subrangetherebetween, including endpoints.

II. Compositions, Devices, & Kits

In an aspect is provided a composition including (a) nucleotidesincluding a free 3′-OH, (b) nucleotides lacking a free 3′-OH, and (c)one or more reagents for decreasing the amount of the nucleotidesincluding a free 3′-OH. In embodiments, the one or more reagents includea depletion primer, a depletion template, and a depletion polymerasethat is active to extend the depletion primer along the depletiontemplate by selectively incorporating the nucleotides including a free3′-OH. In embodiments, the one or more reagents include a depletionprimer and a depletion polymerase that is active to extend the depletionprimer by selectively incorporating the nucleotides including a free3′-OH. In embodiments, the depletion primer is annealed to a depletiontemplate. In embodiments, the depletion primer and the depletiontemplate are portions of a single polynucleotide (e.g., as describedherein). In embodiments, the one or more reagents include a depletionprimer, a depletion template, and a depletion polymerase that is activeto extend the depletion primer along the depletion template byselectively incorporating the nucleotides including a free 3′-OH,wherein the depletion primer and the depletion template are free insolution. In embodiments, the one or more reagents include a depletiontemplate and a depletion polymerase that is active to incorporatenucleotides including a free 3′-OH into the depletion template. Inembodiments, the one or more reagents include one or more nucleotidecyclases active to selectively cyclize the nucleotides including a free3′-OH.

In another aspect is provided a composition including: (a) labelednucleotides including a free 3′-OH, (b) labeled nucleotides lacking afree 3′-OH, and (c) one or more depleting reagents for decreasing theamount of the nucleotides including a free 3′-OH, wherein the one ormore depleting reagents include: (i) one or more depletionpolynucleotides and a depletion polymerase that is active to selectivelyincorporating the nucleotides including a free 3′-OH, wherein thedepletion polynucleotide is free in solution; or (ii) one or morenucleotide cyclases active to selectively cyclize the nucleotidesincluding a free 3′-OH. In embodiments, the composition is stored in asingle container. In embodiments, each nucleotide type (e.g., modifieddATP, dTTP, dCTP, and dGTP) of composition is stored in a differentcontainer with one or more depleting reagents. In embodiments, thecomposition is stored at about 2° C.-8° C., about 20° C.-30° C., orabout 4° C.-37° C. In embodiments, the composition is stored at about 4°C. to about 30° C.

In embodiments, the composition includes nucleotides lacking a free3′-OH. In embodiments, the nucleotides lacking a free 3′-OH include areversible terminator moiety. The reversible terminator moiety may becovalently linked to the 3′-oxygen position of the ribose sugar of anucleotide. For example, a nucleotide lacking a free 3′-OH may berepresented by the formula:

where the nucleobase is adenine or adenine analogue, thymine or thymineanalogue, guanine or guanine analogue, or cytosine or cytosine analogue.

In embodiments, the nucleotide lacking a free 3′-OH has the formula:

wherein B¹ is an optionally substituted nucleobase; R¹ is —OH, amonophosphate moiety, or polyphosphate moiety; R² is —OH or hydrogen;and R³ is a reversible terminator moiety.

In embodiments, B¹ is

In embodiments, B¹ is a divalent nucleobase. In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is -B-L¹⁰⁰-R⁴. B is a divalent cytosine or aderivative thereof, divalent guanine or a derivative thereof, divalentadenine or a derivative thereof, divalent thymine or a derivativethereof, divalent uracil or a derivative thereof, divalent hypoxanthineor a derivative thereof, divalent xanthine or a derivative thereof,divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof. L¹⁰⁰ is a divalent linker; and R⁴ is a detectablemoiety. In embodiments, L¹⁰⁰ is independently a bioconjugate linker, acleavable linker, or a self-immolative linker.

In embodiments, R⁴ is a detectable moiety. In embodiments, R⁴ is afluorescent dye moiety. In embodiments, R⁴ is a detectable moietydescribed herein (e.g., Table 1). In embodiments, R⁴ is a detectablemoiety described in Table 1. In embodiments, R⁴ is a fluorescent dyemoiety wherein the maximum emission of the fluorescent dye moiety isgreater than about 530, 540, or 550 nm. In embodiments, R⁴ is afluorescent dye moiety wherein the maximum emission of the fluorescentdye moiety is greater than 530 nm. In embodiments, R⁴ is a fluorescentdye moiety wherein the maximum emission of the fluorescent dye moiety isless than about 700, 690, or 680 nm. In embodiments, R⁴ is a fluorescentdye moiety wherein the maximum emission of the fluorescent dye moiety isless than 680 nm. In embodiments, R⁴ is a fluorescent dye moiety whereinthe maximum emission of the fluorescent dye moiety is greater than about530 and less than about 680 nm. In embodiments, R⁴ is a fluorescent dyemoiety wherein the maximum emission of the fluorescent dye moiety isgreater than 530 and less than 680 nm. For example, R⁴ may be anyfluorescent moiety described in US Publication 2020/0216682, which isincorporated herein by reference. In embodiments, R⁴ is

TABLE 1 Detectable moieties to be used in selected embodiments.Nucleoside/ nucleotide abbreviation Dye name λmax (nm) dC Atto 532 532dC Atto Rho 6G 535 dC R6G 534 dC Tet 521 dT Atto Rho 11 572 dT Atto 565564 dT Alexa Fluor 568 578 dT dTamra 578 dA Alexa Fluor 647 650 dA Atto647N 644 dA Janelia Fluor 646 646 dG Alexa Fluor 680 682 dG Alexa Fluor700 696 dG CF680R 680

In embodiments, the nucleotides including a free 3′-OH have the formula:

wherein R¹, R², and B¹ are as described herein, including embodiments.

In embodiments the reversible terminator moiety does not decrease thefunction of a polymerase relative to the absence of the reversibleterminator moiety. In embodiments, the reversible terminator moiety doesnot negatively affect DNA polymerase recognition. In embodiments, thereversible terminator moiety does not negatively affect (e.g., limit)the read length of the DNA polymerase. Additional examples of areversible terminator moiety may be found in U.S. Pat. No. 6,664,079, JuJ. et al. (2006) Proc Natl Acad Sci USA 103(52):19635-19640; Ruparel H.et al. (2005) Proc Natl Acad Sci USA 102(17):5932-5937; Wu J. et al.(2007) Proc Natl Acad Sci USA 104(104):16462-16467; Guo J. et al. (2008)Proc Natl Acad Sci USA 105(27): 9145-9150 Bentley D. R. et al. (2008)Nature 456(7218):53-59; or Hutter D. et al. (2010) NucleosidesNucleotides & Nucleic Acids 29:879-895, which are incorporated herein byreference in their entirety for all purposes. In embodiments, areversible terminator moiety includes an azido moiety or a dithiollinking moiety. In embodiments, the reversible terminator moiety is—NH₂, —CN, —CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g.,—CH₂—O—CH₃), methoxyalkenyl (e.g., —CH₂—O—CH═CH₂), or —CH₂N₃. Inembodiments, the reversible terminator moiety comprises a disulfidemoiety. In embodiments, the reversible terminator is a moiety describedin U.S. Pat. No. 10,738,072, which is incorporated herein by referencein its entirety.

In embodiments, the reversible terminator moiety (e.g., represented bythe symbol R³ in Formula (I)) is:

In embodiments, the labeled nucleotide lacking a free 3′-OH has theformula:

R¹ is a polyphosphate moiety, monophosphate moiety, or —OH. R² ishydrogen or —OH. R³ is a reversible terminator moiety. R⁴ is adetectable moiety. B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof. L¹⁰⁰ is adivalent linker.

In embodiments, the method includes the labeled nucleotide including afree 3′-OH has the formula:

R¹ is a polyphosphate moiety, monophosphate moiety, or —OH. R² ishydrogen or —OH. R⁴ is a detectable moiety. B is a divalent cytosine ora derivative thereof, divalent guanine or a derivative thereof, divalentadenine or a derivative thereof, divalent thymine or a derivativethereof, divalent uracil or a derivative thereof, divalent hypoxanthineor a derivative thereof, divalent xanthine or a derivative thereof,divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof. L¹⁰⁰ is a divalent linker.

In embodiments, L¹⁰⁰ is a cleavable linker. The term “cleavable linker”or “cleavable moiety” as used herein refers to a divalent or monovalent,respectively, moiety which is capable of being separated (e.g.,detached, split, disconnected, hydrolyzed, a stable bond within themoiety is broken) into distinct entities. In embodiments, a cleavablelinker is cleavable (e.g., specifically cleavable) in response toexternal stimuli (e.g., enzymes, nucleophilic/basic reagents, reducingagents, photo-irradiation, electrophilic/acidic reagents, organometallicand metal reagents, or oxidizing reagents). In embodiments, a cleavablelinker is a self-immolative linker, a trivalent linker, or a linkercapable of dendritic amplication of signal, or a self-immolativedendrimer containing linker (e.g., all as described in US 2007/0009980,US 2006/0003383, and US 2009/0047699, which are incorporated byreference in their entirety for any purpose). A chemically cleavablelinker refers to a linker which is capable of being split in response tothe presence of a chemical (e.g., acid, base, oxidizing agent, reducingagent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid,fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄),hydrazine (N₂H₄)). A chemically cleavable linker is non-enzymaticallycleavable. In embodiments, the cleavable linker is cleaved by contactingthe cleavable linker with a cleaving agent. In embodiments, the cleavingagent is sodium dithionite (Na₂S₂O₄), weak acid, hydrazine (N₂H₄),Pd(0), or light-irradiation (e.g., ultraviolet radiation). Inembodiments, cleaving includes removing. In embodiments, L¹⁰⁰ includes acleavable site. A “cleavable site” or “scissile linkage” in the contextof a polynucleotide is a site which allows controlled cleavage of thepolynucleotide strand (e.g., the linker, the primer, or thepolynucleotide) by chemical, enzymatic, or photochemical means known inthe art and described herein. A scissile site may refer to the linkageof a nucleotide between two other nucleotides in a nucleotide strand(i.e., an internucleosidic linkage). In embodiments, the scissilelinkage can be located at any position within the one or more nucleicacid molecules, including at or near a terminal end (e.g., the 3′ end ofan oligonucleotide) or in an interior portion of the one or more nucleicacid molecules. In embodiments, conditions suitable for separating ascissile linkage include a modulating the pH and/or the temperature. Inembodiments, a scissile site can include at least one acid-labilelinkage. For example, an acid-labile linkage may include aphosphoramidate linkage. In embodiments, a phosphoramidate linkage canbe hydrolysable under acidic conditions, including mild acidicconditions such as trifluoroacetic acid and a suitable temperature(e.g., 30° C.), or other conditions known in the art, for exampleMatthias Mag, et al Tetrahedron Letters, Volume 33, Issue 48, 1992,7319-7322. In embodiments, the scissile site can include at least onephotolabile internucleosidic linkage (e.g., o-nitrobenzyl linkages, asdescribed in Walker et al, J. Am. Chem. Soc. 1988, 110, 21, 7170-7177),such as o-nitrobenzyloxymethyl or p-nitrobenzyloxymethyl group(s). Inembodiments, the scissile site includes at least one uracil nucleobase.In embodiments, a uracil nucleobase can be cleaved with a uracil DNAglycosylase (UDG) or Formamidopyrimidine DNA Glycosylase Fpg. Inembodiments, the scissile linkage site includes a sequence-specificnicking site having a nucleotide sequence that is recognized and nickedby a nicking endonuclease enzyme or a uracil DNA glycosylase. The term“self-immolative” referring to a linker is used in accordance with itswell understood meaning in Chemistry and Biology as used in US2007/0009980, US 2006/0003383, and US 2009/0047699, which areincorporated by reference in their entirety for any purpose. Inembodiments “self-immolative” referring to a linker refers to a linkerthat is capable of additional cleavage following initial cleavage by anexternal stimuli. The term dendrimer is used in accordance with its wellunderstood meaning in Chemistry. In embodiments, the term“self-immolative dendrimer” is used as described in US 2007/0009980, US2006/0003383, and US 2009/0047699, which are incorporated by referencein their entirety for any purpose and in embodiments refers to adendrimer that is capable of releasing all of its tail units through aself-immolative fragmentation following initial cleavage by an externalstimulus. In embodiments, the cleavable linker is a linker described inU.S. Pat. No. 10,822,653 or U.S. Pat. No. 10,738,072, which isincorporated herein by reference in its entirety.

A “photocleavable linker” (e.g., including or consisting of ano-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of a reducing agent (e.g.,tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker (Binaulda S., et al., Chem. Commun., 2013, 49,2082-2102; Shenoi R. A., et al., J. Am. Chem. Soc., 2012, 134,14945-14957), an azo linker (Rathod, K. M., et al., Chem. Sci. Tran.,2013, 2, 25-28; Leriche G., et al., Eur. J. Org. Chem., 2010, 23,4360-64), an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refer to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent and the agent that cleaves each cleavable linker is different. Inembodiments, an orthogonally is a cleavable linker that followingcleavage the two separated entities (e.g., fluorescent dye, bioconjugatereactive group) do not further react and form a new orthogonallycleavable linker.

In embodiments, L¹⁰⁰ is a cleavable linker comprising an azido moiety, adisulfide moiety, or an alkoxyalkyl moiety. In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

Methods for cleaving the disulfide bond of —S—SO₃H bonds are known inthe art, see for example Meguro et al. Tetrahedron Letters 61 (2020):152198, which is incorporated herein by reference in its entirety. Inembodiments, the cleaving agent is aqueous sodium sulfide (Na₂S). Inembodiments, the cleaving agent is TCEP or THPP.

In embodiments, R¹ is a triphosphate moiety.

In embodiments, R² is hydrogen.

In embodiments, the reversible terminator includes an azido moiety, adisulfide moiety, or an alkoxyalkyl moiety. In embodiments, thereversible terminator moiety is

In embodiments, the nucleotides lacking a free 3′-OH include adetectable label. Typical modified nucleotides attach a uniquedetectable label to the specific location of the base using a cleavablelinker and capping the 3′-OH group with a small reversible-terminatingmoiety so they are still recognized by DNA polymerase as substrates.Examples of detectable labels include imaging agents, includingfluorescent and luminescent substances, molecules, or compositions,including, but not limited to, a variety of organic or inorganic smallmolecules commonly referred to as “dyes,” “labels,” or “indicators.”Examples of fluorophores that may be included in the compounds andcompositions described herein include fluorescent proteins, xanthenederivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, or Texasred), cyanine and derivatives (e.g., cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, or merocyanine), napththalenederivatives (e.g., dansyl or prodan derivatives), coumarin andderivatives, oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole), anthracene derivatives (e.g.,anthraquinones, DRAQ5, DRAQ7, or CyTRAK Orange), pyrene derivatives(e.g., cascade blue and derivatives), oxazine derivatives (e.g., Nilered, Nile blue, cresyl violet, or oxazine 170), acridine derivatives(e.g., proflavin, acridine orange, acridine yellow), arylmethinederivatives (e.g., auramine, crystal violet, or malachite green),tetrapyrrole derivatives (e.g., porphin, phthalocyanine, bilirubin), CFDye™, DRAQ™, CyTRAK™, BODIPY™, Alexa Fluor™, DyLight Fluor™, Atto™,Tracy™, FluoProbes™, Abberior Dyes™, DY™ dyes, MegaStokes Dyes™, SulfoCy™, Seta™ dyes, SeTau™ dyes, Square Dyes™, Quasar™ dyes, Cal Fluor™dyes, SureLight Dyes™, PerCP™, Phycobilisomes™, APC™, APCXL™, RPE™,and/or BPE™. In embodiments, the detectable label is a label in Table 1.The emission from the fluorophores can be detected by any number ofmethods, including but not limited to, fluorescence spectroscopy,fluorescence microscopy, fluorimeters, fluorescent plate readers,infrared scanner analysis, laser scanning confocal microscopy, automatedconfocal nanoscanning, laser spectrophotometers, fluorescent-activatedcell sorters (FACS), image-based analyzers and fluorescent scanners(e.g., gel/membrane scanners). In embodiments, the fluorophore is anaromatic (e.g., polyaromatic) moiety having a conjugated π-electronsystem. In embodiments, the detectable label is a fluorescent dyecapable of exchanging energy with another fluorescent dye (e.g.,fluorescence resonance energy transfer (FRET) chromophores).

In embodiments, the nucleotides lacking a free 3′-OH include a pluralityof different nucleotides that are differently labeled. For example, thecomposition may include a plurality of nucleotide analogues covalentlylinked (e.g., covalently linked with a cleavable linker) to a first dye;a plurality of nucleotide analogues covalently linked (e.g., covalentlylinked with a cleavable linker) to a second dye; a plurality ofnucleotide analogues covalently linked (e.g., covalently linked with acleavable linker) to a third dye; a plurality of nucleotide analoguescovalently linked (e.g., covalently linked with a cleavable linker) to afourth dye; wherein each dye is spectrally distinct from each other. Inembodiments, the composition includes a plurality of adenine or adenineanalogues covalently linked (e.g., covalently linked with a cleavablelinker) to a first dye; a plurality of thymine or thymine analoguescovalently linked (e.g., covalently linked with a cleavable linker) to asecond dye; a plurality of guanine or guanine analogues covalentlylinked (e.g., covalently linked with a cleavable linker) to a third dye;a plurality of cytosine or cytosine analogues covalently linked (e.g.,covalently linked with a cleavable linker) to a fourth dye; wherein eachdye is spectrally distinct from each other. Alternatively, thecomposition may be a two-color sequencing solution and contains only twodye types; see for example the compositions described in U.S. Pat. Nos.9,222,132 and 9,453,258.

In embodiments, the composition further includes a sequencing primer, atarget polynucleotide, and a sequencing polymerase, wherein thesequencing polymerase is active to extend the sequencing primer alongthe target polynucleotide by incorporating one of the nucleotideslacking a free 3′-OH. In embodiments, the sequencing polymerase iscapable of incorporating nucleotides including a free 3′-OH andnucleotides lacking a free 3′-OH. In embodiments, the sequencingpolymerase is mutated to favor incorporating nucleotides lacking a free3′-OH over nucleotides including a free 3′-OH. In embodiments, thesequencing primer hybridizes to the target polynucleotide (e.g., aportion of the target polynucleotide complementary to the sequencingprimer).

In embodiments a source nucleic acid (e.g., genomic template DNA) istreated to form target polynucleotide linear fragments (e.g., about 50to 600 nucleotides). Treatment typically entails fragmentation, such asby chemical fragmentation, enzymatic fragmentation, or mechanicalfragmentation, followed by denaturation to produce single stranded DNAfragments. In embodiments, the target polynucleotide includes anadapter. The adapter may have other functional elements includingtagging sequences (i.e., a barcode), attachment sequences, palindromicsequences, restriction sites, sequencing primer binding sites,functionalization sequences, and the like. Barcodes can be any of avariety of lengths. In embodiments, the adapter includes a barcode thatis 10-50, 20-30, or 4-12 nucleotides in length. In embodiments, theadapter includes a primer binding sequence that is complementary to atleast a portion of the sequencing primer. Primer binding sites can be ofany suitable length. In embodiments, a primer binding site is about orat least about 10, 15, 20, 25, 30, or more nucleotides in length. Inembodiments, a primer binding site is 10-50, 15-30, or 20-25 nucleotidesin length.

In embodiments, the composition includes a plurality of depletiontemplates. In embodiments, the depletion template includes a homopolymersequence. In embodiments, the homopolymer sequence includes consecutiveidentical nucleotides (e.g., a 5-mer of C nucleotides). In embodiments,the homopolymer sequence includes 10 to 30 consecutive identicalnucleotides. In embodiments, the homopolymer sequence includes 2 to 20consecutive identical nucleotides. In embodiments, the homopolymersequence includes 5 to 10 consecutive identical nucleotides. Inembodiments, the homopolymer sequence includes poly (dA), poly (dT),poly (dC), poly (dG), or poly (dU) nucleotides. A depletion template canalso include repeat sequences. Repeat sequences can be any of a varietyof lengths including, for example, 2, 5, 10, 20, 30, 40, 50, 100, 250,500, 1000 nucleotides or more. Repeat sequences can be repeated, eithercontiguously or non-contiguously, any of a variety of times including,for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 times or more.

In embodiments, the depletion primer and the depletion template areportions of a single polynucleotide. In embodiments, the depletionprimer and the depletion template are portions of a singlepolynucleotide including a loop structure. As used herein, the term“loop region” or “loop” refers to a region of a single polynucleotidethat is between sequences of the depletion primer and the depletiontemplate, and remains single-stranded when depletion primer anddepletion template are hybridized to one another. In embodiments, theloop includes about 10 to about 20 random nucleotides. In embodiments,the loop includes a modified nucleotide (e.g., a nucleotide linked to anaffinity tag) to facilitate pull-down or purification methods. Inembodiments, the loop includes a biotinylated nucleotide (e.g.,biotin-11-cytidine-5′-triphosphate). In embodiments, the depletionprimer and the depletion template are portions of a singlepolynucleotide including a hairpin structure. In embodiments, thedepletion primer and the depletion template are portions of a singlepolynucleotide including a hairpin structure and a 5′ overhang. Inembodiments, the depletion polymerase (e.g., TdT) is capable ofincorporating nucleotides to the 3′-OH of a single polynucleotide, andthus only a depletion template is needed. In embodiments, the one ormore depletion polynucleotides includes a depletion primer annealed to adepletion template.

In embodiments, the composition includes the depletion polymerase, andthe nucleotides lacking a free 3′-OH include a modification that blocksstrand incorporation by the depletion polymerase. In embodiments, thedepletion polymerase is different than the sequencing polymerase. Inembodiments, the depletion polymerase shares the same enzymatic backboneas the sequencing enzyme, however the depletion polymerase differs inthe number or placement of amino acid mutations that may be present inthe sequencing enzyme. For example, the depletion enzyme may be a nativepolymerase (e.g., a wild type P. abyssi enzyme) and the sequencingenzyme is a mutated polymerase such as, for example, a mutant P. abyssipolymerase described in WO 2018/148723 or WO 2020/056044, both of whichare incorporated by reference herein.

In embodiments, the depletion polymerase is active at a temperature ofabout 2° C.-65° C., about 2° C.-10° C., or about 4° C.-37° C. Inembodiments, the depletion polymerase is active at about 4° C. Inembodiments, the depletion polymerase is active at about 37° C. Inembodiments, the depletion polymerase is active at about 42° C. Inembodiments, the depletion polymerase is not thermostable above 65° C.In embodiments, the depletion polymerase is not thermostable above 55°C. In embodiments, the depletion polymerase is not thermostable above50° C. In embodiments, the depletion polymerase is not thermostableabove 45° C. In embodiments, the depletion polymerase is notthermostable above 40° C. In embodiments, the depletion polymerase isactive at a temperature of about 20° C.-40° C. In embodiments, thedepletion polymerase is active at about 20° C. One having skill in theart would understand methods and protocols for evaluating enzymaticactivity.

In embodiments, the depletion polymerase includes a Klenow fragment(e.g., Klenow (3′→5′ exo-)) polymerase. In embodiments, the depletionpolymerase is a Klenow fragment polymerase. In embodiments, thedepletion polymerase is a Klenow polymerase. In embodiments, thedepletion polymerase is a Klentaq polymerase. In embodiments, thedepletion polymerase lacks exonuclease activity. In embodiments, thedepletion polymerase is a Klenow Fragment (3′→5′ exo-), which is anN-terminal truncation of DNA Polymerase I which retains polymeraseactivity, but has lost the 5′→3′ exonuclease activity and has mutations(D355A, E357A) which removes the 3′→5′ exonuclease activity. Inembodiments, the depletion polymerase includes a mutant Klenow fragment.Mutant Klenow fragments have been described in the protein sequence ofDNA polymerases I. For example, U.S. Pat. No. 6,329,178 mentions DNApolymerase mutants with altered catalytic activity in which there weremutations in the A motif (the highly conserved sequence DYSQIELR (SEQ IDNO:7), which is incorporated herein by reference in its entirerty).Additionally, Minnick, T. et al., J. Biol. Chem. 274, 3067-3075 (1999),describe a wide variety of E. coli DNA polymerase I (Klenow fragment)mutants in which alanine exchanges have been performed. “Klenowfragment” as used herein means any C-terminal fragment of a family A DNApolymerase which has polymerase activity but no 5′-3′ exonucleaseactivity. In embodiments, additional mutations may be introduced toremove 5′-3′ exonuclease activity. In embodiments, the depletionpolymerase is a Klenow fragment or mutant thereof, soluble guanylylcyclase or mutant thereof, or a terminal deoxynucleotidyl transferase(TdT). In embodiments, the depletion polymerase is a polymeraseincluding an amino acid sequence that is at least 80% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 5, at least onemutation at amino acid position 32 or an amino acid positionfunctionally equivalent to amino acid position 32; a mutation at aminoacid position 34 or an amino acid position functionally equivalent toamino acid position 34; or a mutation at amino acid position 584 or anamino acid position functionally equivalent to amino acid position 584.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 5, at least one mutation at amino acid position 32 oran amino acid position functionally equivalent to amino acid position32; a mutation at amino acid position 34 or an amino acid positionfunctionally equivalent to amino acid position 34; or a mutation atamino acid position 584 or an amino acid position functionallyequivalent to amino acid position 584.

In embodiments, the polymerase is exo-/exo-variant (i.e., does notinclude 3′-5′ or 5′-3′ exonuclease activity). Examples of mutationsgiving rise to an exo/exo-variants include mutations at positions in aparent polymerase corresponding to positions in SEQ ID NO: 5 identifiedas follows: 32 and 34. In embodiments, the polymerase includes a valine,threonine, glycine, or alanine at amino acid position 32. Inembodiments, the polymerase includes a valine at amino acid position 32.In embodiments, the polymerase includes a threonine at amino acidposition 32. In embodiments, the polymerase includes a glycine at aminoacid position 32. In embodiments, the polymerase includes an alanine atamino acid position 32. In embodiments, the polymerase includes a serineat amino acid position 32. In embodiments, the polymerase includes avaline, threonine, glycine, or alanine at amino acid position 34. Inembodiments, the polymerase includes a valine at amino acid position 34.In embodiments, the polymerase includes a threonine at amino acidposition 34. In embodiments, the polymerase includes a glycine at aminoacid position 34. In embodiments, the polymerase includes an alanine atamino acid position 34. In embodiments, the polymerase includes a serineat amino acid position 34.

In embodiments, the polymerase includes an amino acid substitution atposition 584. The amino acid substitution at position 584 may be aserine, glycine, threonine, asparagine, or alanine substitution. Theamino acid substitution at position 584 may be a serine substitution. Inembodiments, the substitution at position 584 includes a polar aminoacid (e.g., threonine, asparagine, or glutamine). In embodiments, theamino acid substitution at position 584 is a selenocysteine. Inembodiments, the substitution at position 584 includes a serine at aminoacid position 584. In embodiments, the substitution at position 584includes a glycine at amino acid position 584. In embodiments, thesubstitution at position 584 includes a threonine at amino acid position584. In embodiments, the substitution at position 584 includes anasparagine at amino acid position 584. In embodiments, the substitutionat position 584 includes an alanine at amino acid position 584.

In embodiments, the nucleotide cyclase is a soluble guanylyl cyclase(also known as guanyl cyclase, guanylyl cyclase, or GC). In embodiments,the cyclase is soluble guanylyl cyclase (e.g., soluble guanylyl cyclaseα1β1, as described in Beste et al Biochemistry. 2012; 51(1):194-204),which has both purinyl and pyrimidinyl cyclase activity and can serve tocyclize all potential nucleotides present in a nucleotide solution(e.g., A, C, G, T/U).

In embodiments, the composition is in a sequencing flow cell. Flow cellsprovide a convenient format for housing an array of clusters produced bythe methods described herein, in particular when subjected to an SBS orother detection technique that involves repeated delivery of reagents incycles. For example, to initiate a first SBS cycle, one or more labelednucleotides and a DNA polymerase in a buffer can be flowed into/througha flow cell that houses an array of clusters. The clusters of an arraywhere primer extension causes a labeled nucleotide to be incorporatedcan then be detected. Optionally, the nucleotides can further include areversible termination moiety that temporarily halts further primerextension once a nucleotide has been added to a primer. For example, anucleotide analog having a reversible terminator moiety can be added toa primer such that subsequent extension cannot occur until a deblockingagent (e.g., a reducing agent) is delivered to remove the moiety. Thus,for embodiments that use reversible termination, a deblocking reagent(e.g., a reducing agent) can be delivered to the flow cell (before,during, or after detection occurs). Washes can be carried out betweenthe various delivery steps as needed. The cycle can then be repeated Ntimes to extend the primer by N nucleotides, thereby detecting asequence of length N. Example SBS procedures, fluidic systems anddetection platforms that can be readily adapted for use with an arrayproduced by the methods of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), US 2018/0274024, WO2017/205336, US 2018/0258472, each of which are incorporated herein intheir entirety for all purposes.

In an aspect is provided a kit. The kit includes one or more of thecompositions as described herein. In embodiments, the includes one ormore DNA polymerases. In embodiments, the kit includes additionalcomponents, such as one or more primers, modified and/or unmodifieddeoxynucleotide triphosphates (dNTPs), buffers, quantification reagents,e.g., intercalating reagents, or reagents binding to the minor groove,(e.g., PicoGreen (Molecular Probes), SybrGreen (Molecular Probes),ethidium bromide, Gelstar (Cambrex) and Vista Green (Amersham)). Inembodiments, the individual components of the kit can be alternativelycontained either together in one storage container or separately in twoor more storage containers (e.g., separate bottles or vials).

In embodiments, the kit includes nucleotides in a buffer. Inembodiments, the kit includes a buffer. For example, the sequencingsolution and/or the chase solution may include a buffer such asethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, acarbonate salt, a phosphate salt, a borate salt,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine (TEMED), andN,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.For example, the buffer may Tris-HCl (pH 9.2 at 25° C.), ammoniumsulfate, MgCl₂, 0.1% Tween® 20, and dNTPs.

In an aspect is provided a microfluidic device for sequencing a targetpolynucleotide. In embodiments, the microfluidic device includes areaction vessel for receiving a composition as described herein; one ormore reservoirs including the composition as described herein; flowpaths from each reservoir to the reaction vessel; and a fluidicscontroller that controls the flow from the reservoir to the reactionvessel.

In an aspect is provided a microfluidic device that includes a heatingelement (e.g., surface heater, such as a thin-film surface heater,) incontact with, within close proximity to, or otherwise thermally coupledto a reservoir within a fluidic manifold (e.g., a fluidic system); thereservoir including internal channels, tunnels, pathways, or other meansfor controlling fluid flow. As the reagent (e.g., a composition asdescribed herein) is moved through a reservoir zone, the surface heaterheats the reagent prior to entry into the reaction vessel. Inembodiments, the reservoir is heated and/or maintained at a firsttemperature range. The fluid system may store fluids for washing orcleaning the fluidic network of the microfluidic device, and also fordiluting the reactants. For example, the fluid system may includevarious reservoirs to store reagents, enzymes, other biomolecules,buffer solutions, aqueous, and non-polar solutions. Furthermore, thefluid system may also include waste reservoirs for receiving wasteproducts. As used herein, fluids may be liquids, gels, gases, or amixture of thereof. Also, a fluid can be a mixture of two or morefluids. The fluidic network may include a plurality of microfluidiccomponents (e.g., fluid lines, pumps, flow cells or other fluidicdevices, manifolds, reservoirs) configured to have one or more fluidsflowing therethrough.

In another embodiment, the microfluidic device includes a heated tubethat increases the temperature of the composition as it transits fromthe reservoir to the reaction vessel. In embodiments, the heatingelement is a heated tube. The tube may be rigid (i.e., fixed) orflexible. In embodiments, a wire is wrapped on the tube and then it iscovered with insulation material (e.g., Kapton, polymer, steel wire orsilicone). In embodiments, the heating element is a nickel inductiveheater. A heating element that includes nickel may be selected as theinduction heating element in the microfluidic device because of therelatively small influence of geometries and faster thermal response. Aheating element provides heat (e.g., an increase in temperature). Inembodiments, the microfluidic device is a nucleic acid sequencingdevice, which refers to an integrated system of one or more chambers,ports, and channels that are interconnected and in fluid communicationand designed for carrying out an analytical reaction or process, eitheralone or in cooperation with an appliance or instrument that providessupport functions, such as sample introduction, fluid and/or reagentdriving means, temperature control, detection systems, data collectionand/or integration systems, for the purpose of determining the nucleicacid sequence of a template polynucleotide. Nucleic acid sequencingdevices may further include valves, pumps, and specialized functionalcoatings on interior walls.

To reduce the thermal gradients that arise in a heated reaction vesselat a given temperature, T_(r×n), when introducing a composition at alower temperature, i.e., T_(composition)<T_(r×n), the composition withinthe heated tube is heated before the composition is introduced into theheated reaction vessel. The particular dimensions of the tube can be abalance of (i) the distance between reservoir containing the unheatedsolution and the reaction vessel; (ii) the desired or required flowrate; (iii) the available pressure differential (ΔP); and (iv) therequired temperature differential (ΔT). Thus, the dimensions of the tubecan be specific to the instrument requirements rather than some uniquecombination that achieves efficient heating.

Controlling the temperature may be carried out by a variety of means.For example, in embodiments, the temperature regulation apparatus is athermoelectric temperature controller, e.g., a Peltier heater/cooler.Alternatively, the temperature regulation apparatus may incorporate aseries of channels through which is flowed a recirculating temperaturecontrolled fluid, e.g., water, ethylene glycol or oil, which is heatedor cooled to a desired temperature, e.g., in an attached water bath. Byway of example, some sequencing by synthesis methods include variouscycles of extension, ligation, cleavage, and/or hybridization in whichit may be desired to cycle the temperature. Further, in some sequencingtechniques, temperatures may range from about 0° C. to about 20° C., toa higher temperature ranging from about 50° C. to about 95° C. fordenaturation and/or other reaction stages.

In embodiments, the microfluidic device includes an imaging system ordetection apparatus. Any of a variety of detection apparatus can beconfigured to detect the reaction vessel or solid support where reagentsinteract. Examples include luminescence detectors, surface plasmonresonance detectors and others known in the art. Exemplary systemshaving fluidic and detection components that can be readily modified foruse in a system herein include, but are not limited to, those set forthin U.S. Pat. Nos. 8,241,573, 8,039,817; or US Pat. App. Pub. No.2012/0270305 A1, each of which is incorporated herein by reference. Inembodiments, the microfluidic device further includes one or moreexcitation lasers. In embodiments, the microfluidic device is a nucleicacid sequencing device. Nucleic acid sequencing devices utilizeexcitation beams to excite labeled nucleotides in the DNA containingsample to enable analysis of the base pairs present within the DNA. Manyof the next-generation sequencing (NGS) technologies use a form ofsequencing by synthesis (SBS), wherein modified nucleotides are usedalong with an enzyme to read the sequence of DNA templates in acontrolled manner. In embodiments, sequencing includes a sequencing bysynthesis event, where individual nucleotides are identified iteratively(e.g., incorporated and detected into a growing complementary strand),as they are polymerized to form a growing complementary strand. Inembodiments, nucleotides added to a growing complementary strand includeboth a label and a reversible chain terminator that prevents furtherextension, such that the nucleotide may be identified by the labelbefore removing the terminator to add and identify a further nucleotide.Such reversible chain terminators include removable 3′ blocking groups,for example as described in U.S. Pat. Nos. 10,738,072, 7,541,444 and7,057,026. Once such a modified nucleotide has been incorporated intothe growing polynucleotide chain complementary to the region of thetemplate being sequenced, there is no free 3′-OH group available todirect further sequence extension and therefore the polymerase cannotadd further nucleotides. Once the identity of the base incorporated intothe growing chain has been determined, the 3′ reversible terminator maybe removed to allow addition of the next successive nucleotide. Inembodiments, the nucleic acid sequencing device utilizes the detectionof four different nucleotides that comprise four different labels.

III. Methods

In an aspect is provided a method of sequencing a target polynucleotide.In embodiments, the method includes (a) incubating the targetpolynucleotide in a composition described herein (e.g., a reactionmixture including a sequencing primer, nucleotides including a free3′-OH, nucleotides lacking a free 3′-OH, and a sequencing polymerase);(b) enzymatically decreasing the amount of the nucleotides including afree 3′-OH; (c) extending the sequencing primer along the targetpolynucleotide using the sequencing polymerase by incorporating one ofthe nucleotides lacking a free 3′-OH; and (d) identifying theincorporated nucleotide. In embodiments, steps (a)-(d) are performed ina sequencing flow cell. In embodiments, steps (c)-(d) are performed in asequencing flow cell, whereas steps (a) and (b) are performed in acontainer (e.g., sequencing cartridge). In embodiments, the targetpolynucleotide is immobilized to a solid substrate.

In an aspect is provided a method of sequencing a target polynucleotide,the method including (a) generating a refined solution by contacting acomposition including a plurality of labeled nucleotides including afree 3′-OH and a plurality of labeled nucleotides lacking a free 3′-OHwith one or more depleting reagents, wherein the one or more depletingreagents include: (i) one or more depletion polynucleotides and adepletion polymerase that is active to selectively incorporate thenucleotides including a free 3′-OH, wherein the depletion polynucleotideis free in solution; or (ii) one or more nucleotide cyclases that isactive to selectively cyclize the nucleotides including a free 3′-OH;(b) inactivating the depletion polymerase or the one or more nucleotidecyclases; (c) contacting a sequencing primer annealed to a targetpolynucleotide with the refined solution and detecting the label of theincorporated labeled nucleotide lacking a free 3′-OH. In embodiments,step (c) is performed in a sequencing flow cell. In embodiments, thetarget polynucleotide is immobilized to a solid substrate. Inembodiments, the method further includes repeating step (c) for aplurality of sequencing cycles. In embodiments, the method includes oneor more wash cycles (e.g., between repeating step (c)). In embodiments,generating a refined solution occurs at a first temperature range ofabout 1° C. to about 45° C. In embodiments, the method includesincreasing the temperature to a second temperature range and reducingthe activity of the depletion polymerase. In embodiments, the depletionpolymerase and the one or more depleting reagents are not removed priorto step (c). In embodiments, the depletion polymerase and the one ormore depleting reagents are present during step (c).

In an aspect is provided a method of decreasing the amount of 3′-OHnucleotide in a sequencing solution, said method including: (a)contacting a sequencing solution with a depleting solution, wherein saidsequencing solution includes a 3′-OH nucleotide and a plurality oflabeled 3′-O-blocked reversible terminator nucleotides and wherein thedepleting solution includes: (i) a depletion polynucleotide and adepletion polymerase, wherein the depletion polymerase incorporates the3′-OH nucleotide into the depletion polynucleotide thereby producing anextended depletion polynucleotide; or (ii) a nucleotide cyclase, whereinthe nucleotide cyclase cyclizes the 3′-OH nucleotide thereby producing acyclized nucleotide; and (b) inactivating the depletion polymerase orthe nucleotide cyclase.

In another aspect is provided a method of depleting labeled nucleotidesincluding a free 3′-OH in a composition including (i) labelednucleotides including a free 3′-OH and (ii) labeled nucleotides lackinga free 3′-OH, the method including: incubating the composition with adepletion polymerase at a first temperature range of about 1° C. toabout 45° C., wherein the depletion polymerase is free in solution andcapable of depleting the labeled nucleotides including a free 3′-OH inthe composition by selectively incorporating the nucleotides including afree 3′-OH into one or more depletion polynucleotides; or selectivelycyclizing the nucleotides including a free 3′-OH with a one or morenucleotide cyclases. In embodiments, the method further includesincorporating one or more labeled nucleotides lacking a free 3′-OH intoa sequencing primer hybridized to a target polynucleotide. Inembodiments, the method further includes detecting the one or morelabeled nucleotides (e.g., detecting the incorporated labelednucleotide).

As used herein, the term “3′-OH nucleotide” refers to a nucleotide withan unblocked hydroxyl, wherein the oxygen atom is at the 3′ position ofthe pentose of the nucleotide. A 3′-OH nucleotide may be incorporatedinto, for example, the 3′ end of a polynucleotide primer by formation ofa phosphodiester bond that results in a DNA extension product. As usedherein, the term “labeled 3′-O-blocked reversible terminatornucleotides” refers to single nucleobases with a labeled 3′-O-blockedreversible terminator that are substrates for sequencing reactions asdescribed herein. In embodiments, the nucleotides including a free 3′-OHhave the formula:

wherein R¹, R², and B¹ are as described herein, including embodiments.In embodiments, the nucleotides including a free 3′-OH have the formula(II) or (II-A).

As used herein, the term “depleting solution” is a solution thatincludes one or more depleting reagents (e.g., one or more depletionpolynucleotides and a depletion polymerase that is active to selectivelyincorporate the nucleotides including a free 3′-OH, wherein thedepletion polynucleotide is free in solution; or one or more nucleotidecyclases that is active to selectively cyclize the nucleotides includinga free 3′-OH) used for reducing the amount of 3′-OH nucleotide in asequencing solution.

As used herein, the term “depletion polynucleotide” refers to apolynucleotide capable of being extended by a depletion polymerase,wherein the depletion polymerase incorporates one or more 3′-OHnucleotide(s). In embodiments, the depletion polynucleotide includes ahomopolymer sequence (e.g., a polyT sequence). In embodiments, thedepletion polynucleotide is a single polynucleotide comprising a hairpinstructure and a 5′ overhang. In embodiments, the depletionpolynucleotides include a depletion primer annealed to a depletiontemplate, wherein the depletion primer has a free 3′-OH. Examples ofdepletion polynucleotides are provided in Table 2. A depletionpolynucleotide may alternatively be referred to herein as a depletionoligonucleotide or depletion oligonucleotide template.

As used herein, the term “depletion polymerase” refers to a polymerasecapable of incorporating 3′-OH nucleotides, an incapable ofincorporating optionally labeled, 3′-O-blocked reversible terminatornucleotides. In embodiments, the depletion polymerase is a polymerasedescribed herein. In embodiments, the depletion polymerase includes aKlenow fragment, or mutant thereof. In embodiments, the depletionpolymerase includes a Klenow fragment. In embodiments, the depletionpolymerase is a Klenow fragment, or a mutant thereof. In embodiments,the depletion polymerase is a bacterial DNA polymerase, eukaryotic DNApolymerase, archaeal DNA polymerase, viral DNA polymerase, or phage DNApolymerases.

As used herein, the term “nucleotide cyclase” refers to an enzymecapable of cyclizing a 3′-OH nucleotide, and incapable of cyclizing anoptionally labeled, 3′-O-blocked reversible terminator nucleotide.

In embodiments, prior to incubating the composition is stored for atleast 1 day, at least 2 days, at least 3 days, or at least 7 days. Inembodiments, prior to incubating the composition is stored for about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about6 weeks, about 7 weeks, or about 8 weeks. In embodiments, prior toincubating the composition is stored for about 1 month, about 2 months,about 3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, or about 12 months. In embodiments, the composition is stored atabout 2° C.-8° C., about 20° C.-30° C., or about 4° C.-37° C.

In embodiments, the method includes inactivating the depletionpolymerase or the one or more nucleotide cyclases includes heatinactivation or chemical inactivation. In embodiments, the methodincludes inactivating the depletion polymerase by increasing thetemperature (e.g., heat inactivation) to reduce and/or eliminate theactivity of the depletion polymerase. For example, many depletionpolymerases are adversely affected by high temperatures and becomedenatured at temperatures above 40° C. In embodiments, the methodincludes denaturing the depletion polymerase. Alternatively, thedepletion polymerase may be rendered inactive via chemical inactivation(e.g., contacting the depletion polymerase with one or more chemicaladditives such as proteinase K, nonionic surfactants, SDS, ordithiothreitol). In embodiments, the method includes inactivating thedepletion polymerase by contacting the depletion polymerase with asurfactant (e.g., sodium dodecyl sulfate, SDS) and a proteinase (e.g.,proteinase K). SDS is a powerful anionic surfactant that at highconcentrations denatures proteins by disturbing the noncovalent bondsthat provide secondary protein structure. In embodiments, the chemicaladditive is dithiothreitol, guanidinium chloride, pronase, Triton X-100,or a combination of one or more of the foregoing additives.

In embodiments, the method includes incubating a composition includingnucleotides including a free 3′-OH, nucleotides lacking a free 3′-OH,and one or more depletion reagents capable of decreasing the amount ofthe nucleotides including a free 3′-OH at a first temperature range(e.g., 1° C. to about 40° C.). In embodiments, the one or more reagentsinclude a depletion polynucleotide (e.g., a double stranded DNAmolecule). In embodiments, the one or more reagents include a depletionpolynucleotide, which includes a depletion primer and a depletiontemplate.

In embodiments, the one or more reagents include a depletion primer, adepletion template, and a depletion polymerase that is active at thefirst temperature range to extend the depletion primer along thedepletion template by selectively incorporating the nucleotidesincluding a free 3′-OH. In embodiments the method further includesincreasing the temperature to a second temperature range (e.g., about40° C. to about 70° C.) to reduce the activity of the depletionreagents. For example, in embodiments, the method includes incubatingthe mixture of nucleotides including a free 3′-OH, nucleotides lacking afree 3′—OH with a depletion primer, a depletion template, and adepletion polymerase at about 1° C. to about 25° C. While incubating atthe first temperature range the amount of nucleotides including a free3′-OH is decreased. Following incubation the temperature is increased toa second temperature range that inactivates the depletion polymerase.For example, temperature may be increased or decreased at a rate ofabout 0.1° C./s to about 5° C./s. In embodiments, temperature may beincreased or decreased at a rate of about 0.1° C./s. In embodiments,temperature may be increased or decreased at a rate of about 0.2° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 0.3° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 0.4° C./s. In embodiments, temperature maybe increased or decreased at a rate of about 0.5° C./s. In embodiments,temperature may be increased or decreased at a rate of about 0.5° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 0.75° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 1° C./s. In embodiments, temperature may beincreased or decreased at a rate of about 1.25° C./s. In embodiments,temperature may be increased or decreased at a rate of about 1.5° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 1.75° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 2° C./s. In embodiments, temperature may beincreased or decreased at a rate of about 2.25° C./s. In embodiments,temperature may be increased or decreased at a rate of about 2.5° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 2.75° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 3° C./s. In embodiments, temperature may beincreased or decreased at a rate of about 3.25° C./s. In embodiments,temperature may be increased or decreased at a rate of about 3.5° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 3.75° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 4° C./s. In embodiments, temperature may beincreased or decreased at a rate of about 4.25° C./s. In embodiments,temperature may be increased or decreased at a rate of about 4.5° C./s.In embodiments, temperature may be increased or decreased at a rate ofabout 4.75° C./s. In embodiments, temperature may be increased ordecreased at a rate of about 5° C./s. For example, a sequencing reactionincludes increasing the reaction temperature to about 55° C. to about65° C.

In embodiments, enzymatically decreasing the amount of the nucleotidesincluding a free 3′-OH includes a depletion polymerase extending adepletion primer along a depletion template by selectively incorporatingthe nucleotides including a free 3′-OH. In embodiments, enzymaticallydecreasing the amount of the nucleotides including a free 3′-OH includesselectively cyclizing the nucleotides including the free 3′-OH using anucleotide cyclase. In embodiments, the nucleotide cyclase is a solubleguanylyl cyclase. In embodiments, the method decreases the amount of thenucleotides including a free 3′-OH relative to a control (e.g., theamount of nucleotides in the absence of the one or more depletionreagents).

In embodiments, the labeled nucleotide lacking a free 3′-OH has theformula:

R¹ is a polyphosphate moiety, monophosphate moiety, or —OH. R² ishydrogen or —OH. R³ is a reversible terminator moiety. R⁴ is adetectable moiety. B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof. L¹⁰⁰ is adivalent linker.

In embodiments, the method includes the labeled nucleotide including afree 3′-OH has the formula:

R¹ is a polyphosphate moiety, monophosphate moiety, or —OH. R² ishydrogen or —OH. R⁴ is a detectable moiety. B is a divalent cytosine ora derivative thereof, divalent guanine or a derivative thereof, divalentadenine or a derivative thereof, divalent thymine or a derivativethereof, divalent uracil or a derivative thereof, divalent hypoxanthineor a derivative thereof, divalent xanthine or a derivative thereof,divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof. L¹⁰⁰ is a divalent linker.

In embodiments, L¹⁰⁰ is a cleavable linker. In embodiments, L¹⁰⁰ is acleavable linker comprising an azido moiety, a disulfide moiety, or analkoxyalkyl moiety.

In embodiments, R¹ is a triphosphate moiety.

In embodiments, R² is hydrogen. In embodiments, R² is —OH.

In embodiments, the reversible terminator includes an azido moiety, adisulfide moiety, or an alkoxyalkyl moiety.

In embodiments, the nucleotides lacking a free 3′-OH include areversible terminator moiety (e.g., a reversible terminator moiety asdescribed herein, including embodiments). In embodiments, the reversibleterminator moiety is:

In embodiments, the reversible terminator moiety is

In embodiments, the nucleotides lacking a free 3′-OH include adetectable label. In embodiments, the nucleotides lacking a free 3′-OHinclude a plurality of different nucleotides that are differentlylabeled. The modified nucleotides may carry a label (e.g., a fluorescentlabel) to facilitate their detection. Each nucleotide type may carry adifferent fluorescent label. However, the detectable label need not be afluorescent label. Any label can be used which allows the detection ofan incorporated nucleotide. One method for detecting fluorescentlylabeled nucleotides includes using laser light of a wavelength specificfor the labeled nucleotides, or the use of other suitable sources ofillumination. The fluorescence from the label on the nucleotide may bedetected (e.g., by a CCD camera or other suitable detection means).

In embodiments, the depletion template includes a homopolymer sequence.In embodiments, the homopolymer sequence includes consecutive identicalnucleotides (e.g., a 5-mer of C nucleotides). In embodiments, thehomopolymer sequence includes 10 to 30 consecutive identicalnucleotides. In embodiments, the homopolymer sequence includes 2 to 20consecutive identical nucleotides. In embodiments, the homopolymersequence includes 5 to 10 consecutive identical nucleotides. Inembodiments, the homopolymer sequence includes poly (dA), poly (dT),poly (dC), poly (dG), or poly (dU) nucleotides. A depletion template canalso include repeat sequences. Repeat sequences can be any of a varietyof lengths including, for example, 2, 5, 10, 20, 30, 40, 50, 100, 250,500, 1000 nucleotides or more. Repeat sequences can be repeated, eithercontiguously or non-contiguously, any of a variety of times including,for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 times or more.

In embodiments, the depletion primer and the depletion template areportions of a single polynucleotide. In embodiments, the depletionprimer and the depletion template are portions of a singlepolynucleotide including a loop structure. As used herein, the term“loop region” or “loop” refers to a region of a single polynucleotidethat is between sequences of the depletion primer and the depletiontemplate, and remains single-stranded when the depletion primer anddepletion template are hybridized to one another. In embodiments, theloop includes about 10 to about 20 random nucleotides. In embodiments,the loop includes a modified nucleotide (e.g., a nucleotide linked to anaffinity tag) to facilitate pull-down or purification methods. Inembodiments, the loop includes a biotinylated nucleotide (e.g.,biotin-11-cytidine-5′-triphosphate). In embodiments, the depletionprimer and the depletion template are portions of a singlepolynucleotide including a hairpin structure. In embodiments, thedepletion primer and the depletion template are portions of a singlepolynucleotide including a hairpin structure and a 5′ overhang.

In embodiments, the nucleotides lacking a free 3′-OH include amodification that blocks strand incorporation by the depletionpolymerase (e.g., the nucleotides include a reversible terminatormoiety).

In embodiments, the depletion polymerase is active at a temperature ofabout 2° C.-65° C., about 2° C.-10° C., or about 4° C.-37° C. Inembodiments, the depletion polymerase is active at about 4° C. Inembodiments, the depletion polymerase is active at about 37° C. Inembodiments, the depletion polymerase is active at about 42° C. Inembodiments, the depletion polymerase is not active during step c). Inembodiments, the depletion polymerase is not thermostable above 65° C.In embodiments, the depletion polymerase is active at a temperature ofabout 20° C.-40° C. In embodiments, the depletion polymerase is activeat about 20° C.

In embodiments, the depletion polymerase is a polymerase describedherein. In embodiments, the depletion polymerase includes a Klenowfragment, or mutant thereof. In embodiments, the depletion polymeraseincludes a Klenow fragment. In embodiments, the depletion polymerase isa Klenow fragment, or a mutant thereof. In embodiments, the depletionpolymerase is a bacterial DNA polymerase, eukaryotic DNA polymerase,archaeal DNA polymerase, viral DNA polymerase, or phage DNA polymerases.Bacterial DNA polymerases include E. coli DNA polymerases I, II and III,IV and V, the Klenow fragment of E. coli DNA polymerase, Clostridiumstercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNApolymerase and Sulfolobus solfataricus (Sso) DNA polymerase. EukaryoticDNA polymerases include DNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ,and k, as well as the Revl polymerase (terminal deoxycytidyltransferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNApolymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1,phi-29-like DNA polymerases, PZA DNA polymerase, phi-15 DNA polymerase,Cpl DNA polymerase, Cpl DNA polymerase, T7 DNA polymerase, and T4polymerase. Other useful DNA polymerases include thermostable and/orthermophilic DNA polymerases such as Thermus aquaticus (Taq) DNApolymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcuszilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase,Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNApolymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp.GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD)DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNApolymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcusacidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase;Thermococcus sp. go N-7 DNA polymerase; Pyrodictium occultum DNApolymerase; Methanococcus voltae DNA polymerase; Methanococcusthermoautotrophicum DNA polymerase; Methanococcus jannaschii DNApolymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol);Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase;Pyrococcus islandicunm DNA polymerase; Thermococcus fumicolans DNApolymerase; Aeropyrunm pemix DNA polymerase; and the heterodimeric DNApolymerase DP1/DP2. In embodiments, the polymerase is 3PDX polymerase asdisclosed in U.S. Pat. No. 8,703,461, the disclosure of which isincorporated herein by reference. In embodiments, the polymerase is areverse transcriptase. Exemplary reverse transcriptases include, but arenot limited to, HIV-1 reverse transcriptase from human immunodeficiencyvirus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from humanimmunodeficiency virus type 2, M-MLV reverse transcriptase from theMoloney murine leukemia virus, AMV reverse transcriptase from the avianmyeloblastosis virus, or Telomerase reverse transcriptase. Inembodiments, the depletion polymerase is a nucleotide cyclase. Inembodiments, the depletion polymerase is a terminal transferase (e.g.,terminal deoxycytidyl transferase or terminal deoxynucleotidyltransferase (TdT)). In embodiments, the depletion polymerase is an RNAdependent polymerase. In embodiments, the depletion polymerase is aKlenow fragment or mutant thereof, soluble guanylyl cyclase or mutantthereof, or a terminal deoxynucleotidyl transferase (TdT).

In embodiments, the depletion polymerase is active at a temperature ofabout 1° C. to about 45° C. In embodiments, the depletion polymerase isactive at a temperature of about 10° C. to about 40° C. In embodiments,the depletion polymerase is active at a temperature of about 4° C. toabout 37° C. In embodiments, the depletion polymerase is not activeabove a temperature of about 45° C. (e.g., the thermostable polymerasedoes not have substantial measurable activity).

In embodiments, the target polynucleotide is within a cluster ofamplicons. In embodiments, the target polynucleotide is an amplicon(i.e., the amplification product of a source nucleic acid). Inembodiments, prior to step c) the method includes an amplificationmethod. Suitable methods for amplification include, but are not limitedto, the polymerase chain reaction (PCR), strand displacementamplification (SDA), transcription mediated amplification (TMA) andnucleic acid sequence based amplification (NASBA), for example, asdescribed in U.S. Pat. No. 8,003,354, which is incorporated herein byreference in its entirety. The above amplification methods can beemployed to amplify one or more nucleic acids of interest. For example,PCR, multiplex PCR, SDA, TMA, NASBA and the like can be utilized toamplify immobilized nucleic acid fragments. In embodiments,amplification includes thermal bridge polymerase chain reactionamplification; for example, as exemplified by the disclosures of U.S.Pat. Nos. 5,641,658; 7,115,400; 7,790,418; U.S. Patent Publ. No.2008/0009420, each of which is incorporated herein by reference in itsentirety. In general, bridge amplification uses repeated steps ofannealing of primers to templates, primer extension, and separation ofextended primers from templates. Because the forward and reverse primersare attached to the solid substrate, the extension products releasedupon separation from an initial template are also attached to the solidsupport. Both strands are immobilized on the solid substrate at the 5′end, preferably via a covalent attachment. The 3′ end of anamplification product is then permitted to anneal to a nearby reverseprimer, forming a “bridge” structure. The reverse primer is thenextended to produce a further template molecule that can form anotherbridge. During bridge PCR, additional chemical additives may be includedin the reaction mixture, in which the DNA strands are denatured byflowing a denaturant over the DNA, which chemically denaturescomplementary strands. This is followed by washing out the denaturantand reintroducing an amplification polymerase in buffer conditions thatallow primer annealing and extension.

In embodiments, the amplifying includes rolling circle amplification(RCA) or rolling circle transcription (RCT) (see, e.g., Lizardi et al.,Nat. Genet. 19:225-232 (1998), which is incorporated herein by referencein its entirety). Several suitable rolling circle amplification methodsare known in the art. For example, RCA amplifies a circularpolynucleotide (e.g., DNA) by polymerase extension of an amplificationprimer complementary to a portion of the input polynucleotide. Thisprocess generates copies of the circular polynucleotide template suchthat multiple complements of the template sequence arranged end to endin tandem are generated (i.e., a concatemer) locally preserved at thesite of the circle formation. In embodiments, amplifying occurs atisothermal conditions. In embodiments, amplifying includes hybridizationchain reaction (HCR). HCR uses a pair of complementary, kineticallytrapped hairpin oligomers to propagate a chain reaction of hybridizationevents, as described in Dirks, R. M., & Pierce, N. A. (2004) PNAS USA,101(43), 15275-15278, which is incorporated herein by reference for allpurposes. In embodiments, the amplifying includes branched rollingcircle amplification (BRCA); e.g., as described in Fan T, Mao Y, Sun Q,et al. Cancer Sci. 2018; 109:2897-2906, which is incorporated herein byreference in its entirety. In embodiments, the amplifying includeshyberbranched rolling circle amplification (HRCA). Hyperbranched RCAuses a second primer complementary to the first amplification product.This allows products to be replicated by a strand-displacementmechanism, which yields drastic amplification within an isothermalreaction (Lage et al., Genome Research 13:294-307 (2003), which isincorporated herein by reference in its entirety). In embodiments,amplifying includes polymerase extension of an amplification primer withan amplification polymerase.

In embodiments, the sequencing polymerase is a Taq polymerase,Therminator γ, 9° N polymerase (exo-), Therminator II, Therminator III,or Therminator IX. In embodiments, the sequencing polymerase isTherminator γ. In embodiments, the sequencing polymerase is 9° Npolymerase (exo-). In embodiments, the sequencing polymerase isTherminator II. In embodiments, the sequencing polymerase is TherminatorIII. In embodiments, the sequencing polymerase is Therminator IX. Inembodiments, the sequencing polymerase is a Taq polymerase. Inembodiments, the sequencing polymerase is a sequencing polymerase. Inembodiments, the sequencing polymerase is 9° N and mutants thereof. Inembodiments, the sequencing polymerase is Phi29 and mutants thereof. Inembodiments, the DNA polymerase is a modified archaeal DNA polymerase.In embodiments, the polymerase is a reverse transcriptase. Inembodiments, the polymerase is a mutant P. abyssi polymerase (e.g., suchas a mutant P. abyssi polymerase described in WO 2018/148723 or WO2020/056044, both of which are incorporated by reference herein). Inembodiments, the polymerase is DNA polymerase, a terminaldeoxynucleotidyl transferase, or a reverse transcriptase. Inembodiments, the enzyme is a DNA polymerase, such as DNA polymerase 812(Pol 812) or DNA polymerase 1901 (Pol 1901), e.g., a polymerasedescribed in US 2020/0131484, and US 2020/0181587, both of which areincorporated by reference herein.

In embodiments, the sequencing polymerase is a bacterial DNA polymerase,eukaryotic DNA polymerase, archaeal DNA polymerase, viral DNApolymerase, or phage DNA polymerases. Bacterial DNA polymerases includeE. coli DNA polymerases I, II and III, IV and V, the Klenow fragment ofE. coli DNA polymerase, Clostridium stercorariun (Cst) DNA polymerase,Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and k, as well as the Revlpolymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cpl DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNApolymerases include thermostable and/or thermophilic DNA polymerasessuch as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrunm pemix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. In embodiments, thepolymerase is 3PDX polymerase as disclosed in U.S. Pat. No. 8,703,461,the disclosure of which is incorporated herein by reference. Inembodiments, the polymerase is a reverse transcriptase. Exemplaryreverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, or Telomerasereverse transcriptase.

A variety of sequencing methodologies can be used such as sequencing-bysynthesis (SBS), pyrosequencing, sequencing by ligation (SBL), orsequencing by hybridization (SBH). In SBS, extension of a nucleic acidprimer along a nucleic acid template is monitored to determine thesequence of nucleotides in the template. The underlying chemical processcan be catalyzed by a polymerase, wherein fluorescently labelednucleotides are added to a primer (thereby extending the primer) in atemplate dependent fashion such that detection of the order and type ofnucleotides added to the primer can be used to determine the sequence ofthe template. A plurality of different nucleic acid fragments that havebeen attached at different locations of an array can be subjected to anSBS technique under conditions where events occurring for differenttemplates can be distinguished due to their location in the array. Inembodiments, the sequencing step includes annealing and extending asequencing primer to incorporate a detectable label that indicates theidentity of a nucleotide in the target polynucleotide, detecting thedetectable label, and repeating the extending and detecting of steps. Inembodiments, the methods include sequencing one or more bases of atarget polynucleotide by extending a sequencing primer hybridized to atarget polynucleotide. In embodiments, the sequencing step may beaccomplished by a sequencing-by-synthesis (SBS) process. In embodiments,sequencing comprises a sequencing by synthesis process, where individualnucleotides are identified iteratively, as they are polymerized to forma growing complementary strand. In embodiments, nucleotides added to agrowing complementary strand include both a label and a reversible chainterminator that prevents further extension, such that the nucleotide maybe identified by the label before removing the terminator to add andidentify a further nucleotide. Such reversible chain terminators includeremovable 3′ blocking groups, for example as described in U.S. Pat. Nos.7,541,444 and 7,057,026. Once such a modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced, there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. Once the identity of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template. Sequencing canbe carried out using any suitable sequencing-by-synthesis (SBS)technique, wherein modified nucleotides are added successively to a free3′ hydroxyl group, typically initially provided by a sequencing primer,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. In embodiments, sequencing includes detecting a sequence ofsignals. In embodiments, sequencing includes extension of a sequencingprimer with labeled nucleotides. Examples of sequencing include, but arenot limited to, sequencing by synthesis (SBS) processes in whichreversibly terminated nucleotides carrying fluorescent dyes areincorporated into a growing strand, complementary to the target strandbeing sequenced. In embodiments, the nucleotides are labeled with up tofour unique fluorescent dyes. In embodiments, the nucleotides arelabeled with at least two unique fluorescent dyes. In embodiments, thereadout is accomplished by epifluorescence imaging.

In embodiments, sequencing includes a plurality of sequencing cycles. Inembodiments, sequencing includes 20 to 100 sequencing cycles. Inembodiments, sequencing includes 50 to 100 sequencing cycles. Inembodiments, sequencing includes 50 to 300 sequencing cycles. Inembodiments, sequencing includes 150 to 300 sequencing cycles. Inembodiments, sequencing includes 200 to 300 sequencing cycles. Inembodiments, sequencing includes 200 to 300 sequencing cycles. Inembodiments, sequencing includes 50 to 500 sequencing cycles. Inembodiments, sequencing includes 100 to 1000 sequencing cycles. Inembodiments, sequencing includes 50 to 150 sequencing cycles. Inembodiments, sequencing includes at least 10, 20, 30, 40, or 50sequencing cycles. In embodiments, sequencing includes at least 50, 60,70, 80, 90, or 100 sequencing cycles. In embodiments, sequencingincludes at least 10 sequencing cycles. In embodiments, sequencingincludes 10 to 20 sequencing cycles. In embodiments, sequencing includes10, 11, 12, 13, 14, or 15 sequencing cycles. In embodiments, sequencingincludes (a) extending a sequencing primer by incorporating a labelednucleotide, or labeled nucleotide analogue and (b) detecting the labelto generate a signal for each incorporated nucleotide or nucleotideanalogue.

In embodiments, sequencing includes extending a sequencing primer togenerate a sequencing read. In embodiments, sequencing includesextending a sequencing primer by incorporating a labeled nucleotide, orlabeled nucleotide analogue and detecting the label to generate a signalfor each incorporated nucleotide or nucleotide analogue. In embodiments,the labeled nucleotide or labeled nucleotide analogue includes areversible terminator moiety. In embodiments, the method includesrepeating the cycle of extending a sequencing primer with a labelednucleotide analogue containing a reversible terminator moiety, detectingthe labeled nucleotide analogue and removing the reversible terminatormoiety and detectable label. In embodiments, the method includes one ormore wash steps between each cycle to facilitate removal of the labeland reversible terminator moiety from the reaction vessel (e.g., flowcell).

Use of the sequencing method outlined above is a non-limiting example,as essentially any sequencing methodology which relies on successiveincorporation of nucleotides into a polynucleotide chain can be used.Suitable alternative techniques include, for example, pyrosequencingmethods, FISSEQ (fluorescent in situ sequencing), MPSS (massivelyparallel signature sequencing), or sequencing by ligation-based methods.

In an aspect is provided a method of increasing storage stability(alternatively referred to as shelf-life) of modified nucleotides. Inembodiments, the modified nucleotides are for use in a sequencingreaction. In embodiments, the method of increasing the storage stabilityincludes (a) storing the modified nucleotides in solution at about 2°C.-65° C. for at least 12 hours, wherein the modified nucleotidesinclude nucleotides lacking a free 3′-OH, and wherein the solutionincludes nucleotides including a free 3′-OH; and (b) depleting thenucleotides including a free 3′-OH during storage. In embodiments,depleting the nucleotides including a free 3′-OH during storage includesextending a depletion primer along a depletion template using adepletion polymerase that selectively incorporates the nucleotidesincluding a free 3′-OH, wherein the depletion primer and the depletiontemplate are free in solution. In embodiments, depleting the nucleotidesincluding a free 3′-OH during storage includes selectively cyclizing thenucleotides including the free 3′-OH using a nucleotide cyclase. Inembodiments, the method of increasing storage stability of modifiednucleotides is measured relative to a control (e.g., modifiednucleotides not subjected to depleting the nucleotides including a free3′-OH during storage). In embodiments, any of the various components ofthe solution (e.g., the nucleotides, primer, template, polymerase,and/or nucleotide cyclase) are as described herein, such as with regardto any of the various aspects disclosed herein.

In embodiments, the method of increasing the storage stability includes(a) storing the modified nucleotides in solution at about 1° C.-40° C.for one or more minutes, wherein the modified nucleotides includenucleotides lacking a free 3′-OH, and wherein the solution includesnucleotides including a free 3′-OH; and (b) depleting the nucleotidesincluding a free 3′-OH during storage. In embodiments, step (a) includesmaintaining the modified nucleotides in solution at about 15° C.-30° C.for one or more minutes. In embodiments, step (a) includes maintainingthe modified nucleotides in solution at about 15° C.-30° C. for 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more minutes. In embodiments, step (a) includesmaintaining the modified nucleotides in solution at about 15° C.-30° C.for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours.

In embodiments, the storing is for at least 1 day, 2 days, 3 days, or 7days. In embodiments, the storing is for between at least 1 day to about2 days. In embodiments, the storing is for at least 1 day to about 3days. In embodiments, the storing is for at least 1 day to about 7 days.In embodiments, the storing is for more than 7 days. In embodiments, thestoring is for more than 1 month.

In embodiments, the storing is at about 2° C.-8° C., about 20° C.-30°C., or about 4° C.-37° C. In embodiments, the storing is at about 2°C.-8° C. In embodiments, the storing is at about 20° C.-30° C. Inembodiments, the storing is at about 4° C.-37° C. In embodiments, thestoring is at about 2° C. In embodiments, the storing is at about 4° C.In embodiments, the storing is at about 8° C. In embodiments, thestoring is at about 20° C. In embodiments, the storing is at about 30°C. In embodiments, the storing is at about 37° C.

In embodiments, the storing is at about 2° C.-8° C. for at least 1 day.In embodiments, the storing is at about 2° C. for at least 1 day. Inembodiments, the storing is at about 4° C. for at least 1 day. Inembodiments, the storing is at about 8° C. for at least 1 day.

In embodiments, the storing is at about 20° C.-30° C. for at least 1day. In embodiments, the storing is at about 20° C. for at least 1 day.In embodiments, the storing is at about 25° C. for at least 1 day. Inembodiments, the storing is at about 25° C. for at least 1, 2, 3, 4, 5or more days. In embodiments, the storing is at about 25° C. for atleast 1 day. In embodiments, the storing is at about 25° C. for at least1 day. In embodiments, the storing is at about 25° C. for at least 1, 2,3, 4, 5 or more weeks. In embodiments, the storing is at about 25° C.for at least 1, 2, 3, 4, 5 or more months. In embodiments, the storingis at about 30° C. for at least 1 day.

In an aspect is provided a method of increasing the shelf life of acomposition including modified nucleotides, the method including: (a)storing the composition as described herein at about 1° C. to about 40°C. for one or more minutes; and (b) depleting the labeled nucleotidesincluding a free 3′-OH during the storing, wherein the depletingincludes: (i) incorporating with a depleting polymerase the nucleotidesincluding a free 3′-OH into one or more depletion polynucleotides insolution; or (ii) selectively cyclizing the nucleotides including thefree 3′-OH using a nucleotide cyclase; wherein depleting the nucleotidesincluding a free 3′-OH increases the shelf life of the kit includingmodified nucleotides relative to a control (e.g., the same kit withoutcontacting the composition one or more depleting reagents).

In embodiments, the method of increasing storage stability (i.e., theshelf life) of modified nucleotides further includes sequencing a targetpolynucleotide in a reaction mixture, wherein the reaction mixtureincludes the target polynucleotide, a sequencing primer, a sequencingpolymerase, and at least a portion of the stored solution of modifiednucleotides.

In embodiments, the portion of the stored solution of modifiednucleotides is an unfractionated portion of the stored solution.

In an aspect is provided a method of decreasing one or more sequencingerrors in a plurality of sequencing cycles, the method including (a)contacting a composition including a plurality of labeled nucleotidesincluding a free 3′-OH and a plurality of labeled nucleotides lacking afree 3′-OH with one or more depleting reagents to generate a refinedsolution, wherein the one or more depleting reagents include: (i) adepletion polynucleotide and a depletion polymerase that is active toselectively incorporating the nucleotides including a free 3′-OH,wherein the depletion polynucleotide is free in solution; or (ii) one ormore nucleotide cyclases that is active to selectively cyclize thenucleotides including a free 3′-OH; (b) inactivating the depletionpolymerase or the one or more nucleotide cyclases; (c) contacting asequencing primer annealed to a target polynucleotide with the refinedsolution and detecting the label of the incorporated labeled nucleotidelacking a free 3′-OH; and repeating step (c), wherein the sequencingerrors is reduced relative to a control (e.g., the same compositionwithout contacting one or more depleting reagents). In embodiments, theone or more sequencing errors includes a carry forward error. Inembodiments, the carry forward error is reduced relative to a control.

In embodiments, the carry forward error is less than 0.2% for theplurality of sequencing cycles (e.g., 100, 150, 200, 250, 300, or moresequencing cycles). In embodiments, the carry forward error is less than0.15% for the plurality of sequencing cycles (e.g., 100, 150, 200, 250,300, or more sequencing cycles). In embodiments, the carry forward erroris less than 0.1% for the plurality of sequencing cycles (e.g., 100,150, 200, 250, 300, or more sequencing cycles). In embodiments, thecarry forward error is less than 0.05% for the plurality of sequencingcycles (e.g., 100, 150, 200, 250, 300, or more sequencing cycles). Inembodiments, the carry forward error is 0.2% for the plurality ofsequencing cycles (e.g., 100, 150, 200, 250, 300, or more sequencingcycles). In embodiments, the carry forward error is 0.15% for theplurality of sequencing cycles (e.g., 100, 150, 200, 250, 300, or moresequencing cycles). In embodiments, the carry forward error is 0.1% forthe plurality of sequencing cycles (e.g., 100, 150, 200, 250, 300, ormore sequencing cycles). In embodiments, the carry forward error is0.05% for the plurality of sequencing cycles (e.g., 100, 150, 200, 250,300, or more sequencing cycles). In embodiments, the carry forward erroris not greater than 0.2% for the plurality of sequencing cycles (e.g.,100, 150, 200, 250, 300, or more sequencing cycles). In embodiments, thecarry forward error is not greater than 0.15% for the plurality ofsequencing cycles (e.g., 100, 150, 200, 250, 300, or more sequencingcycles). In embodiments, the carry forward error is not greater than0.1% for the plurality of sequencing cycles (e.g., 100, 150, 200, 250,300, or more sequencing cycles). In embodiments, the carry forward erroris not greater than 0.05% for the plurality of sequencing cycles (e.g.,100, 150, 200, 250, 300, or more sequencing cycles).

EXAMPLES Example 1. Depleting Nucleotide Impurities

Sequencing-by-synthesis (SBS) methodologies employ serial incorporationand detection of labeled nucleotide analogues. For example,high-throughput SBS technology uses cleavable fluorescent nucleotidereversible terminator (NRT) sequencing chemistry. Nucleotides (e.g., A,C, G, T, and/or U) are modified by attaching a unique cleavablefluorophore to the specific location of the nucleobase and capping the3′-OH group of the nucleotide sugar with a small reversible moiety (alsoreferred to herein as a reversible terminator) so that they are stillrecognized by DNA polymerase as substrates. The reversible terminatortemporarily halts the polymerase reaction after nucleotide incorporationwhile the fluorophore signal is detected. After incorporation and signaldetection, the fluorophore and the reversible terminator are cleaved toresume the polymerase reaction in the next cycle. Ensemble-based SBSincludes sequencing collections of identical sequences (i.e., monoclonalclusters of amplicons) and determining their sequence by synthesis ofthe complement in a stepwise, synchronous fashion. This results in anaverage sequence signal from all the amplicons present in a cluster perincorporation event.

A challenge of using reversible terminators in NGS technologies is thepresence of impurities, such as natural nucleotides or non-reversibleterminator-containing nucleotides. DNA polymerases generallydiscriminate against modified nucleotides in favor of 3′-OH bearingnucleotide counterparts when presented as a mixture. This typicallyleads to the clusters of monoclonal amplicons being out-of-phase,reducing sequencing accuracy and limiting sequencing read lengths. Longread lengths require an effective solution to the synchrony problems inensemble-based SBS. One such phase loss effect relates to an “incompleteextension” (IE) event or error (also referred to herein as a “lagerror”). An IE event may occur as a result of a failure of a sequencingreaction to incorporate one or more nucleotide species into one or morenascent molecules for a given extension round of the sequence, forexample, which may result in subsequent reactions being at a sequenceposition that is out of phase with the sequence position for themajority of the population (e.g., certain template extensions fallbehind the main template population). IE events may arise, for example,due of a lack of nucleotide availability to a portion of thetemplate/polymerase complexes of a population. Alternatively, or inaddition, IE events may be caused by a defective or absent polymerase,or an incorporated nucleotide that does not have a 3′ OH available(e.g., retains a reversible terminator) for nucleotide polymerization.

Another such phase loss effect relates to a “carry forward” (CF) eventor error (also referred to herein as a “lead error”). A CF event mayoccur as a result of an improper additional extension of a nascentmolecule by incorporation of one or more nucleotide species in asequence or strand position that is ahead and thus out of phase with thesequence or strand position of the rest of the population. CF events mayarise, for example, because of the misincorporation of a nucleotidespecies, or in certain instances, because of contamination fromnucleotides remaining from a previous cycle (e.g., which may result froman insufficient or incomplete washing of the reaction chamber). Forexample, a small fraction of a “dT” nucleotide cycle may be present orcarry forward to a “dC” nucleotide cycle. The presence of bothnucleotides may lead to an undesirable extension of a fraction of thegrowing strands where the “dT” nucleotide is incorporated in addition tothe “dC” nucleotide such that multiple different nucleotideincorporations events take place where only a single type of nucleotideincorporation would normally be expected. Alternatively, some strandsmay extend faster when the reversible terminator of the nucleotide to beincorporated is removed prematurely, or the solution of reversiblyterminated nucleotides contains impurities (e.g., natural nucleotides ormodified nucleotides bearing a 3′ hydroxyl group). CF events may alsoarise because of a polymerase error (e.g., there may be an improperincorporation of a nucleotide species into the nascent molecule that isnot complementary to the nucleotide species on the template molecule).

Errors or phasing issues related to IE and CF events (alternativelyreferred to as phasing and/or prephasing errors) may be exacerbated overtime because of the accumulation of such events, which may causedegradation of sequence signal or quality over time and an overallreduction in the practical read length of the system (e.g., the numberof nucleotides that can be sequenced for a given template). The presentdisclosure reflects the discovery that sequencing performance (e.g.,efficiency and/or accuracy of sequencing) may be improved by utilizingthe methods described herein.

Sequencing by synthesis of nucleic acids ideally requires the controlled(i.e. one at a time), yet rapid, incorporation of the correctcomplementary nucleotide opposite the oligonucleotide being sequenced.For example, using nucleotides bearing a 3′ reversible terminator allowsfor successive nucleotides to be incorporated into a polynucleotidechain in a controlled manner. Following detection, the removal of thereversible terminator leaves a free 3′ hydroxyl group for addition ofthe next nucleotide (see FIG. 1 ).

Typically, many polynucleotides are confined to an area of a discreteregion (referred to as a cluster) and are synchronized in theirnucleotide incorporation and detection. For example, at the start of asequencing reaction, after hybridization of the sequencing primer, 100%of the strands within the cluster are synchronized. As the strands areextended, individual strands may fall behind or extend faster than themajority of the strands. This loss of synchronization is amplified asthe number of sequencing rounds increases and eventually, the backgroundnoise from the unsynchronized strands becomes too great to accuratelycall the correct base. Some strands may extend faster when thereversible terminator of the nucleotide to be incorporated is removedprematurely, or the solution of reversibly terminated nucleotidescontains impurities (e.g., natural nucleotides or modified nucleotidesbearing a 3′ hydroxyl group), resulting in the clusters of monoclonalamplicons being out-of-phase. For example, see FIG. 2 for an overview ofthe undesired process.

Without a reversible terminator present on the nucleotide, an additionalnucleotide is capable of being incorporated and detected, resulting indephasing from surrounding amplicons in the cluster. Thisasynchronization event results in a lower quality individual base callsand less accurate sequencing reads. The nucleotide solutions and methodsdescribed herein results in faster SBS cycle times, lower out-of-phasevalues, and permit longer sequencing read lengths.

Improvements to modified nucleotide stability or methods of removingunterminated nucleotides from nucleotide solutions remains a challenge.Impurities may be present immediately following manufacturing andpurification. Although high-performance liquid chromatography (HPLC) canpurify immediately following manufacturing 3′-O-modified-nucleotides,the quantity of 3′-OH bearing nucleotides remains high enough to causeasynchronization events in sequencing. Additionally, stored nucleotidesolutions may degrade over time, resulting in premature cleaving of thereversible terminator, leading to an increased concentration of 3′-OHbearing nucleotides in a stored nucleotide solution.

An enzymatic mop-up strategy was described in Metzker et al(BioTechniques 25:814-817 (1998)), where streptavidin containing beads,a biotinylated primer and mop-up template is added to a solution. In thepresence of a polymerase, non-terminated nucleotides are incorporatedinto the mop-up template. The beads containing the now incorporatednon-terminated nucleotides are then isolated and discarded. The mop-upstrategy was originally developed for non-labeled 3′-O-modifiednucleotides. In the context of SBS, nucleotides (e.g., A, C, G, T,and/or U) are typically modified by attaching a unique fluorophore tothe nucleobase and capping the 3′-OH group of the nucleotide sugar witha reversible terminator. When following the procedure outlined inMetzker, the fluorophores non-specifically interact with thestreptavidin coated beads, saturating the remaining functional groupsand preventing the beads from reducing the labeled non-terminatednucleotides and limiting the efficacy of this technique. Additionally,separating the beads within a microfluidic device is nontrivial andresulted in coagulated structures arresting fluid flow.

Applicants developed processes to remove non-terminated nucleotides fromsolutions (e.g., nucleotide solutions and sequencing solutions) capableof being used directly in a microfluidic device, such as a sequencingdevice, referred to herein as “live-polishing” or “nucleotide impuritydepletion”. For example, one method to live-polish a nucleotide solutionincludes incubating the solution with a depletion polymerase (e.g.,Klenow (3′→5′ exo-)) and an oligo template (e.g., a depletion template)in solution, depicted in FIGS. 3A-3B. While incubating, thenon-terminated nucleotides (i.e., nucleotides having a 3′-OH moiety) areincorporated into the depletion template. The resulting polishednucleotide solution no longer contains non-terminated nucleotides (asdepicted in FIG. 3B). Because a “live-polished” solution in accordancewith some embodiments can be used directly, it does not require clean-upto remove the depletion polymerase or depletion template. As such, thebeads of Metzker are not needed, and both the depletion primer anddepletion template may instead be free in solution.

Alternatively, the nucleotide solution is incubated with a nucleotidecyclase. For example, incubating a nucleotide solution containingnon-terminated nucleotides with an adenylyl cyclase, guanylyl cyclase,or cytidylyl cyclase converts the non-terminated nucleotides to cyclicmonophosphates, rendering them non-incorporable by a sequencing enzymein a subsequent sequencing reaction. In embodiments, the cyclase issoluble guanylyl cyclase (e.g., soluble guanylyl cyclase α1β1, asdescribed in see Beste et al Biochemistry. 2012; 51(1):194-204), whichhas both purinyl and pyrimidinyl cyclase activity and can serve tocyclize all potential nucleotides present in a nucleotide solution(e.g., A, C, G, T/U).

These depletion protocols are capable of removing non-terminatednucleotides from solutions containing a mixture of reversibly-terminatedmodified nucleotides and non-terminated nucleotides, and can beimplemented immediately after manufacturing and purifying the modifiednucleotides. For example, a nucleotide solution containing the necessarycomponents for live-polishing may then be stored in a vessel (e.g.,stored at 4° C.) and is continually removing non-terminated nucleotidesfrom the solution as they form within the vessel. Live-polishingcontinues while the nucleotide solution is stored until ready to be usedin a microfluidic device, which often is weeks to months followinginitial nucleotide production. Live-polishing may allow an extension ofthe expiration date relative to non-polished nucleotide solutions, andmay also allow expired nucleotide solutions to be salvaged and used in asequencing reaction. The quantity of non-terminated nucleotides isreduced relative to a non live-polished nucleotide solution.

Alternatively, a sequencing solution (e.g., a nucleotide solutioncontained in a commercial kits) may also be live-polished while beingused on a microfluidic device. For example, a commercial kit containinga sequencing solution may be mixed with a depletion enzyme in amicrofluidic device, prior to entry into a flow cell. In embodiments,the solution is mixed with a depletion enzyme while in transit to theflow cell. In embodiments, the solution is mixed with a depletion enzymeand maintained at a suitable reaction temperature, prior to entry intothe flow cell. Importantly, this process can be performed within amicrofluidic device, such as a sequencing instrument such that allcontaminating non-terminated modified nucleotides are removed from thesolution immediately prior to a sequencing event.

The presence of the depletion enzyme does not negatively affect thesequencing quality. Upon entry into the flow cell, which contains anucleic acid to be sequenced and typically includes clusters of nucleicacids to be sequenced, the solution (e.g., nucleotide solution orsequencing solution) contains a depletion enzyme. The reactiontemperatures for a sequencing reaction may inactivate the depletionenzyme. Moreover, the depletion enzyme is selected such that it isincapable of incorporating 3′-O-modified nucleotides so if it is activeat the sequencing reaction temperatures it is not competitive with thesequencing enzyme (i.e., an enzyme which is capable of incorporating3′-O-modified nucleotides).

To establish the depletion protocol, each labeled reversibly-terminatednucleotide (C, T, A, and G) solution is isolated in separate containers.Alternatively, a nucleotide solution containing a mixture of all fourlabeled reversibly-terminated nucleotides with C, T, A, and Gnucleotides may be used. The methods and solutions described herein areapplicable to any reversibly terminated nucleotide, for example, at the3′ position of the nucleotide and may be a chemically cleavable moietysuch as an allyl group, an azidomethyl group or a methoxymethyl group,or may be an enzymnatically cleavable group such as a phosphate ester.For the purposes of this experiment we used reversible terminatednucleotides described in U.S. Pat. No. 10,738,072, which is incorporatedherein by reference for all purposes. To each nucleotide solution, 40 μLof Klenow buffer and 8 μL of a specific depletion oligonucleotidetemplate is added. The sequences of the depletion oligonucleotidetemplates are described in Table 2. The depletion oligonucleotidetemplates are each self-priming hairpins with a 5′-overhang with apoly(N) sequence, where N is T, G, C, or A. For example, the G-capturedepletion oligonucleotide template has a (C)₁₅ 5′-overhang such thatwhen added to a nucleotide solution containing non-terminated (i.e.,free 3′-OH) deoxyguanosine triphosphate (dGTP) nucleotides they areincorporated by the depletion enzyme into the G-capture template; thisis partially illustrated in FIGS. 3A-3B. Alternatively, a primer cananneal to the depletion template. Note, to minimize any secondarystructures and ease depletion oligonucleotide template synthesis for theC-capture depletion oligonucleotide template, the 5′-overhang terminateswith a poly-T tail. Klenow exo- is added to each nucleotide solution andincubated at 37° C. for about 90 minutes. The depleted nucleotidesolutions were then quantified and stored at 4° C.

Oligo name Sequence A-capture 5′-TTTTTTTTTTTTTTTGGAGGTGACAGGTTTTTCCT(SEQ ID  GTCACCTCC-3′ NO: 1) C-capture5′-TTTGGGGGGGGGACGTGACAGGTTTTTCCTGTCAC (SEQ ID  CTCC-3′ NO: 2 G-capture5′-CCCCCCCCCCCCCCCGGAGGTGACAGGTTTTTCCT (SEQ ID  GTCACCTCC-3′ NO: 3)T-capture 5′-AAAAAAAAAAAAAAAGGAGGTGACAGGTTTTTCCT (SEQ ID  GTCACCTCC-3′NO: 4)

Experiments to quantify the lead percentage were carried out todetermine the percent of non-terminated nucleotides in a bulk nucleotidesolution. Performing the lead assay determines whether depletion hasbeen effective in reducing the amount of non-terminated nucleotides. Inthis example, the efficiency of depletion in removing non-terminatednucleotides was assessed using depleted nucleotide solutions (with adye-labeled G nucleotide) stored at either 4° C. or 37° C. for 1 day, 3days, or 1 week to simulate depletion during long-term nucleotidestorage, with fresh Klenow enzyme added at time 0. An additional set oftests were included wherein supplemental depletion oligonucleotidetemplate was added to the depleted nucleotide solution at time 0. Alltests were performed in duplicate.

The lead assay was performed in a streptavidin-coated multi-well plate.Template nucleic acids were bound to streptavidin-coated beads, and thebead-template complexes were then attached to the bottom of each well,with a single template present per well. The lead extension enzyme wasdiluted in incorporation buffer, and 40 uL of the enzyme solution wasadded to each well to pre-bind the enzyme to the template/primer duplex.The lead extension enzyme does not accept modified nucleotides (i.e.,does not incorporate reversibly-terminated nucleotides). The plates werethen incubated for 5 minutes at 65° C. Wells were then washed andimaged.

As described supra, the depleted nucleotide solutions tested were storedat either 4° C. or 37° C. for 1 day, 3 days, or 1 week prior to use inthe lead assay. A nucleotide solution which had not live-polished wasused as a no-storage control. Prior to testing, the nucleotide solutionswere pre-warmed at 55° C. Nucleotide solutions were added to each welland incubated at 55° C. for 10 minutes. The wells were then washed withEDTA Wash Buffer to inactivate the enzyme, and the wells were imaged.

Results for the experiment assessing the effect of depletion on leadover time are summarized in FIG. 4 . Generally, storing the non-depletedmodified nucleotides (reported as (−) Depletion in FIG. 4A) results inapproximately 0.1% lead increase relative to a fresh nucleotidesolution, referred to as “F” in FIG. 4A. Unpolished modified nucleotidesolutions are stable at 4° C. and maintain approximately 0.73% lead for1-day, 3-day, and 7-day old nucleotide solutions. Storing the unpolishedmodified nucleotides at 37° C., however, results in a dramatic increasein lead over time. The lead % increases from 0.72% to 1.25% following aweek of storage at 37° C., as reported in FIG. 4A.

With the depletion protocols as described herein, the lead % issignificantly reduced. For example, storing the polished modifiednucleotides (reported as (+) Depletion in FIG. 4B) at both 4° C. and 37°C. results in approximately 0.19% lead for 1-day, 3-day, and 7-day oldnucleotide solutions. Taken together, these results show a benefit fromlive-polishing on the reduction in non-terminated nucleotides across arange of storage conditions. For a solution containing 2000 modifiednucleotides stored at 37° C. for at least a week without polishing,approximately 25 of the modified nucleotides will not contain areversible terminator. In contrast when live polished nucleotidesolutions are used, a solution containing 2000 modified nucleotidesstored at 37° C. for at least a week, approximately 4 of the modifiednucleotides will not contain a reversible terminator.

Example 2. Depletion Enzyme in a Sequencing Reaction

A 50-cycle sequencing run was conducted in the presence of variousconcentrations of a depletion enzyme. Nucleic acid templates werehybridized to surface immobilized primers. A sequencing solutioncontaining components necessary for sequencing, including a nucleotidesolution containing labeled reversibly-terminated nucleotides, a buffer,salts (e.g., magnesium sulfate, potassium chloride) was mixed with adepletion polymerase, Klenow Fragment (3′→5′ exo-) (NEB Catalog #M0407B)and depletion templates. The concentrations of the depletion polymerasewere varied: 0 μL, 120 μL, and 400 μL of a stock solution containing 5U/μL, corresponding to control, 3× (0.00204 U/ul), and 10× (0.0068 U/ul)depletion enzyme concentrations. Following a 50-cycle sequencingexperiment at different concentrations of a depletion enzyme, nosignificant difference in the quality score was observed (see FIG. 5 ).Taken together with the results presented in Example 1, depletion iseffective at reducing non-terminated nucleotide populations, and thepresence of depletion components (e.g., Klenow enzyme) does not impactsequencing quality during a sequencing reaction.

Modified nucleotides that contain a unique cleavably-linked fluorophoreand a reversible-terminating moiety capping the 3′-OH group, forexample, those described in U.S. 2017/0130051, WO 2017/058953, WO2019/164977, and U.S. Pat. No. 10,738,072, have shown sensitivity tocysteines present in sequencing polymerases. The cysteines normally forma disulfide bridge, however in the presence of sequencing solutions andconditions, the disulfide bridge may break to form two reactive thiols.These thiols may act to prematurely cleave the linker and/or reversibleterminator, acting as a weak reducing agent, increasing asynchronousshifts in sequencing runs that are detrimental to sequencing accuracy.Protocols were adjusted to remove any thiol containing reagents. Forexample, enzymes are commonly stored in DTT (referred to asdithiothreitol and/or Clelands reagent) which is used to stabilizeenzymes and other proteins.

There is a need for a depletion polymerase that has reduced interferencewith the modified nucleotides used in sequencing applications. Providedherein are novel polymerases wherein the cysteine amino acid was mutated(C584S in SEQ ID NO:5). While serine was chosen as an initial mutation,any amino acid that eliminates the ability to form free thiols and doesnot perturb the stability nor function of the polymerase is envisioned(e.g., glycine, threonine, selenocysteine or alanine). Variants lackinga cysteine were capable of incorporating nucleotides, andadvantageously, the remaining modified nucleotides exhibited greaterstability (i.e., did not prematurely deblock or lose the detectablemoiety) relative to a polymerase that contained one or more cysteines.

The wild-type enzyme has the sequence (SEQ ID NO: 5):VISYDNYVTILDEETLKAWIAKLEKAPVFAFDTETDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGQNLKYDRGILANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIERNGVKIDPKVLHNHSEELTLRLAELEKKAHEIAGEEFNLSSTKQLQTILFEKQGIKPLKKTPGGAPSTSELSSTDPNLQNIPVRNEEGRRIRQAFIAPEDYVIVSADYSQIELRIMAHLSRDKGLLTAFAEGKDIHRATAAEVFGLPLETVTSEQRRSAKAINFGLIYGMSAFGLARQLNIPRKEAQKYMDLYFERYPGVLEYMERTRAQAKEQGYVETLDGRRLYLPDIKSSNGARRAAAERAAINAPMQGTAADIIKRAMIAVDAWLQAEQPRVRMIMQVHDELVFEVHKDDVDAVAKQIHQLMENCTRLDVPLLVEVGSGENWDQAH.The in-house mutant includes the sequence (SEQ ID NO: 6):MVISYDNYVTILDEETLKAWIAKLEKAPVFAFATATDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGQNLKYDRGILANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIERNGVKIDPKVLHNHSEELTLRLAELEKKAHEIAGEEFNLSSTKQLQTILFEKQGIKPLKKTPGGAPSTSEEVLEELALDYPLPKVILEYRGLAKLKSTYTDKLPLMINPKTGRVHTSYHQAVTATGRLSSTDPNLQNIPVRNEEGRRIRQAFIAPEDYVIVSADYSQIELRIMAHLSRDKGLLTAFAEGKDIHRATAAEVFGLPLETVTSEQRRSAKAINFGLIYGMSAFGLARQLNIPRKEAQKYMDLYFERYPGVLEYMERTRAQAKEQGYVETLDGRRLYLPDIKSSNGARRAAAERAAINAPMQGTAADIIKRAMIAVDAWLQAEQPRVRMIMQVHDELVFEVHKDDVDAVAKQIHQLMENSTRLDVPLLVEVGSGENWDQAH.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

P-Embodiments

The present disclosure provides the following illustrative embodiments.

Embodiment P1. A composition comprising: (a) nucleotides comprising afree 3′-OH, (b) nucleotides lacking a free 3′-OH, and (c) one or morereagents for decreasing the amount of the nucleotides comprising a free3′-OH, wherein the one or more reagents comprise: (i) a depletionprimer, a depletion template, and a depletion polymerase that is activeto extend the depletion primer along the depletion template byselectively incorporating the nucleotides comprising a free 3′-OH,wherein the depletion primer and the depletion template are free insolution; or (ii) one or more nucleotide cyclases active to selectivelycyclize the nucleotides comprising a free 3′-OH.

Embodiment P2. The composition of Embodiment P1, wherein the nucleotideslacking a free 3′-OH comprise a reversible terminator moiety.

Embodiment P3. The composition of Embodiment P1 or Embodiment P2,wherein the nucleotides lacking a free 3′-OH comprise a detectablelabel.

Embodiment P4. The composition of Embodiment P3, wherein the nucleotideslacking a free 3′-OH comprise a plurality of different nucleotides thatare differently labeled.

Embodiment P5. The composition of any one of Embodiment P1-EmbodimentP4, wherein the depletion template comprises a homopolymer sequence.

Embodiment P6. The composition of any one of Embodiment P1-EmbodimentP5, wherein the depletion primer and the depletion template are portionsof a single polynucleotide comprising a hairpin structure and a 5′overhang.

Embodiment P7. The composition of any one of Embodiment P1-EmbodimentP6, wherein the composition comprises the depletion polymerase, and thenucleotides lacking a free 3′-OH comprise a modification that blocksstrand incorporation by the depletion polymerase.

Embodiment P8. The composition of any one of Embodiment P1-EmbodimentP7, wherein the depletion polymerase is active at a temperature of about2° C.-65° C., about 2° C.-10° C., or about 4° C.-37° C.

Embodiment P9. The composition of any one of Embodiment P1-EmbodimentP8, wherein the depletion polymerase is not thermostable above 65° C.

Embodiment P10. The composition of any one of Embodiment P1-EmbodimentP9, wherein the depletion polymerase comprises a Klenow fragment.

Embodiment P11. The composition of any one of Embodiment P1-EmbodimentP10, further comprising a sequencing primer, a target polynucleotide,and a sequencing polymerase, wherein the sequencing polymerase is activeto extend the sequencing primer along the target polynucleotide byincorporating one of the nucleotides lacking a free 3′-OH.

Embodiment P12. The composition of any one of Embodiment P1-EmbodimentP11, wherein the composition is in a sequencing flow cell.

Embodiment P13. The composition of any one of Embodiment P1-EmbodimentP4, Embodiment P11, or Embodiment P12, wherein the nucleotide cyclase isa soluble guanylyl cyclase.

Embodiment P14. A method of sequencing a target polynucleotide, themethod comprising: (a) incubating the target polynucleotide in areaction mixture comprising a sequencing primer, nucleotides comprisinga free 3′-OH, nucleotides lacking a free 3′-OH, and a sequencingpolymerase; (b) enzymatically decreasing the amount of the nucleotidescomprising a free 3′-OH; (c) extending the sequencing primer along thetarget polynucleotide using the sequencing polymerase by incorporatingone of the nucleotides lacking a free 3′-OH; and (d) identifying theincorporated nucleotide.

Embodiment P15. The method of Embodiment P14, wherein enzymaticallydecreasing the amount of the nucleotides comprising a free 3′-OHcomprises a depletion polymerase extending a depletion primer along adepletion template by selectively incorporating the nucleotidescomprising a free 3′-OH.

Embodiment P16. The method of Embodiment P14 or Embodiment P15, whereinthe nucleotides lacking a free 3′-OH comprise a reversible terminatormoiety.

Embodiment P17. The method of any one of Embodiment P14-Embodiment P16,wherein the nucleotides lacking a free 3′-OH comprise a detectablelabel.

Embodiment P18. The method of Embodiment P17, wherein the nucleotideslacking a free 3′-OH comprise a plurality of different nucleotides thatare differently labeled.

Embodiment P19. The method of any one of Embodiment P15-Embodiment P18,wherein the depletion template comprises a homopolymer sequence.

Embodiment P20. The method of any one of Embodiment P15-Embodiment P19,wherein the depletion primer and the depletion template are portions ofa single polynucleotide comprising a hairpin structure and a 5′overhang.

Embodiment P21. The method of any one of Embodiment P15-Embodiment P20,wherein the nucleotides lacking a free 3′-OH comprise a modificationthat blocks strand incorporation by the depletion polymerase.

Embodiment P22. The method of any one of Embodiment P15-Embodiment P21,wherein the depletion polymerase is active at a temperature of about 2°C.-65° C., about 2° C.-10° C., or about 4° C.-37° C.

Embodiment P23. The method of any one of Embodiment P15-Embodiment P22,wherein the depletion polymerase is not thermostable above 65° C.

Embodiment P24. The method of any one of Embodiment P15-Embodiment P23,wherein the depletion polymerase comprises a Klenow fragment.

Embodiment P25. The method of any one of Embodiment P14-Embodiment P24,wherein steps (a)-(d) are performed in a sequencing flow cell.

Embodiment P26. The method of any one of Embodiment P14, EmbodimentP16-Embodiment P18, or Embodiment P25, wherein the enzymaticallydecreasing the amount of the nucleotides comprising a free 3′-OHcomprises selectively cyclizing the nucleotides comprising the free3′-OH using a nucleotide cyclase.

Embodiment P27. The method of Embodiment P26, wherein the nucleotidecyclase is a soluble guanylyl cyclase.

Embodiment P28. A method of increasing storage stability of modifiednucleotides for use in a sequencing reaction, the method comprising: (a)storing the modified nucleotides in solution at about 2° C.-65° C. forat least 12 hours, wherein the modified nucleotides comprise nucleotideslacking a free 3′-OH, and wherein the solution comprises nucleotidescomprising a free 3′-OH; and (b) depleting the nucleotides comprising afree 3′-OH during said storing, wherein said depleting comprises: (i)extending a depletion primer along a depletion template using adepletion polymerase that selectively incorporates the nucleotidescomprising a free 3′-OH, wherein the depletion primer and the depletiontemplate are free in solution; or (ii) selectively cyclizing thenucleotides comprising the free 3′-OH using a nucleotide cyclase.

Embodiment P29. The method of Embodiment P28, wherein the nucleotideslacking a free 3′-OH comprise a reversible terminator moiety.

Embodiment P30. The method of Embodiment P28 or Embodiment P29, whereinthe nucleotides lacking a free 3′-OH comprise a detectable label.

Embodiment P31. The method of Embodiment P30, wherein the nucleotideslacking a free 3′-OH comprise a plurality of different nucleotides thatare differently labeled.

Embodiment P32. The method of any one of Embodiment P28-Embodiment P31,wherein the depletion template comprises a homopolymer sequence.

Embodiment P33. The method of any one of Embodiment P28-Embodiment P32,wherein the depletion primer and the depletion template are portions ofa single polynucleotide comprising a hairpin structure and a 5′overhang.

Embodiment P34. The method of any one of Embodiment P28-Embodiment P33,wherein the nucleotides lacking a free 3′-OH comprise a modificationthat blocks strand incorporation by the depletion polymerase.

Embodiment P35. The method of any one of Embodiment P28-Embodiment P34,wherein said storing is at about 2° C.-8° C., about 20° C.-30° C., orabout 4° C.-37° C.

Embodiment P36. The method of any one of Embodiment P28-Embodiment P35,wherein said storing is for at least 1 day, 2 days, 3 days, or 7 days.

Embodiment P37. The method of Embodiment P36 wherein said storing is atabout 2° C.-8° C. for at least 1 day.

Embodiment P38. The method of Embodiment P36 wherein said storing is atabout 20° C.-30° C. for at least 1 day.

Embodiment P39. The method of any one of Embodiment P28-Embodiment P38,wherein the depletion polymerase is not thermostable above 65° C.

Embodiment P40. The method of any one of Embodiment P28-Embodiment P39,wherein the depletion polymerase comprises a Klenow fragment.

Embodiment P41. The method of any one of Embodiment P28-Embodiment P31or Embodiment P35-Embodiment P38, wherein the nucleotide cyclase is asoluble guanylyl cyclase.

Embodiment P42. The method of any one of Embodiment P28-Embodiment P41,the method further comprising sequencing a target polynucleotide in areaction mixture, wherein the reaction mixture comprises the targetpolynucleotide, a sequencing primer, a sequencing polymerase, and atleast a portion of the stored solution of modified nucleotides.

Embodiment P43. The method of Embodiment P42, wherein the portion of thestored solution of modified nucleotides is an unfractionated portion ofthe stored solution.

Additional Embodiments

The present disclosure provides the following additional illustrativeembodiments.

Embodiment 1. A method of sequencing a target polynucleotide, saidmethod comprising: (a) generating a refined solution by contacting acomposition comprising a plurality of labeled nucleotides comprising afree 3′-OH and a plurality of labeled nucleotides lacking a free 3′-OHwith one or more depleting reagents, wherein the one or more depletingreagents comprise: (i) one or more depletion polynucleotides and adepletion polymerase that is active to selectively incorporate thenucleotides comprising a free 3′-OH, wherein the depletionpolynucleotide is free in solution; or (ii) one or more nucleotidecyclases that is active to selectively cyclize the nucleotidescomprising a free 3′-OH; (b) inactivating the depletion polymerase orthe one or more nucleotide cyclases; and (c) contacting a sequencingprimer annealed to a target polynucleotide with the refined solution anddetecting the label of the incorporated labeled nucleotide lacking afree 3′-OH.

Embodiment 2. The method of embodiment 1, further comprising repeatingstep (c).

Embodiment 3. The method of embodiment 1 or 2, wherein inactivating thedepletion polymerase or the one or more nucleotide cyclases comprisesheat inactivation or chemical inactivation.

Embodiment 4. The method of any one of embodiments 1 to 3, wherein thedepletion polymerase comprises a Klenow fragment, or mutant thereof.

Embodiment 5. The method of any one of embodiments 1 to 3, wherein thedepletion polymerase is a Klenow fragment or mutant thereof, solubleguanylyl cyclase or mutant thereof, or a terminal deoxynucleotidyltransferase (TdT).

Embodiment 6. The method of any one of embodiments 1 to 3, wherein thedepletion polymerase is active at a temperature of about 1° C. to about45° C.

Embodiment 7. The method of any one of embodiments 1 to 3, wherein thedepletion polymerase is active at a temperature of about 4° C. to about37° C.

Embodiment 8. The method of any one of embodiments 1 to 3, wherein thedepletion polymerase is not active above a temperature of about 45° C.

Embodiment 9. The method of any one of embodiments 1 to 8, wherein theone or more depletion polynucleotides comprise a homopolymer sequence.

Embodiment 10. The method of any one of embodiments 1 to 8, wherein theone or more depletion polynucleotides comprises a single polynucleotidecomprising a hairpin structure and a 5′ overhang.

Embodiment 11. The method of any one of embodiments 1 to 8, wherein theone or more depletion polynucleotides comprises a depletion primerannealed to a depletion template.

Embodiment 12. The method of any one of embodiments 1 to 11, whereingenerating a refined solution occurs at a first temperature range ofabout ° C. to about 45° C.

Embodiment 13. The method of any one of embodiments 1 to 12, furthercomprising increasing the temperature to a second temperature range andreducing the activity of the depletion polymerase.

Embodiment 14. The method of any one of embodiments 1 to 13, wherein thenucleotides lacking a free 3′-OH comprise a reversible terminatormoiety.

Embodiment 15. The method of any one of embodiments 1 to 14, wherein thelabeled nucleotide lacking a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is hydrogen or —OH; R³ is a reversible terminator moiety; R⁴ is adetectable moiety; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof; and L¹⁰⁰ isa divalent linker.

Embodiment 16. The method of any one of embodiments 1 to 15, wherein thelabeled nucleotide comprising a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is PGP-hydrogen or —OH; R⁴ is a detectable moiety; B is a divalentcytosine or a derivative thereof, divalent guanine or a derivativethereof, divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof; and L¹⁰⁰ is a divalent linker.

Embodiment 17. The method of embodiment 15 or 16, wherein L¹⁰⁰ is acleavable linker.

Embodiment 18. The method of any one of embodiments 15 to 17, wherein R¹is a triphosphate moiety.

Embodiment 19. The method of any one of embodiments 15 to 18, wherein R²is hydrogen.

Embodiment 20. The method of any one of embodiments 14 to 19, whereinthe reversible terminator comprises an azido moiety, a disulfide moiety,or an alkoxyalkyl moiety.

Embodiment 21. A method of depleting labeled nucleotides comprising afree 3′-OH in a composition comprising (i) labeled nucleotidescomprising a free 3′-OH and (ii) labeled nucleotides lacking a free3′-OH, said method comprising: incubating the composition with adepletion polymerase at a first temperature range of about 1° C. toabout 45° C., wherein the depletion polymerase is free in solution andcapable of depleting the labeled nucleotides comprising a free 3′-OH inthe composition by selectively incorporating the nucleotides comprisinga free 3′-OH into one or more depletion polynucleotides; or selectivelycyclizing the nucleotides comprising a free 3′-OH with a one or morenucleotide cyclases.

Embodiment 22. The method of embodiment 21, further comprisingincorporating one or more labeled nucleotides lacking a free 3′-OH intoa sequencing primer hybridized to a target polynucleotide.

Embodiment 23. The method of embodiment 22, further comprising detectingthe one or more labeled nucleotides.

Embodiment 24. The method of any one of embodiments 21 to 23, whereinprior to incubating the composition is stored for at least 1 day, atleast 2 days, at least 3 days, or at least 7 days.

Embodiment 25. The method of any one of embodiments 21 to 23, whereinprior to incubating the composition is stored for about 1 week, about 2weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about7 weeks, or about 8 weeks.

Embodiment 26. The method of any one of embodiments 21 to 23, whereinprior to incubating the composition is stored for about 1 month, about 2months, about 3 months, about 4 months, about 5 months, about 6 months,about 7 months, about 8 months, about 9 months, about 10 months, about11 months, or about 12 months.

Embodiment 27. The method of any one of embodiments 24 to 26, whereinthe composition is stored at about 2° C.-8° C., about 20° C.-30° C., orabout 4° C.-37° C.

Embodiment 28. The method of any one of embodiments 21 to 27, furthercomprising inactivating the depletion polymerase or the one or morenucleotide cyclases.

Embodiment 29. The method of embodiment 28, wherein inactivating thedepletion polymerase or the one or more nucleotide cyclases comprisesheat inactivation or chemical inactivation.

Embodiment 30. The method of any one of embodiments 21 to 29, whereinthe depletion polymerase comprises a Klenow fragment, or mutant thereof.

Embodiment 31. The method of any one of embodiments 21 to 29, whereinthe depletion polymerase is a Klenow fragment or mutant thereof, solubleguanylyl cyclase or mutant thereof, or a terminal deoxynucleotidyltransferase (TdT).

Embodiment 32. The method of any one of embodiments 21 to 29, whereinthe depletion polymerase is active at a temperature of about 1° C. toabout 45° C.

Embodiment 33. The method of any one of embodiments 21 to 29, whereinthe depletion polymerase is active at a temperature of about 4° C. toabout 37° C.

Embodiment 34. The method of any one of embodiments 21 to 29, whereinthe depletion polymerase is not active above a temperature of about 45°C.

Embodiment 35. The method of any one of embodiments 21 to 34, whereinthe one or more depletion polynucleotides comprise a homopolymersequence.

Embodiment 36. The method of any one of embodiments 21 to 34, whereinthe one or more depletion polynucleotides comprises a singlepolynucleotide comprising a hairpin structure and a 5′ overhang.

Embodiment 37. The method of any one of embodiments 21 to 34, whereinthe depletion polynucleotide comprises depletion primer annealed to adepletion template.

Embodiment 38. The method of any one of embodiments 21 to 37, furthercomprising increasing the temperature to a second temperature range andreducing the activity of the depletion enzyme.

Embodiment 39. The method of any one of embodiments 21 to 38, whereinthe nucleotides lacking a free 3′-OH comprise a reversible terminatormoiety.

Embodiment 40. The method of any one of embodiments 21 to 39, whereinthe labeled nucleotide lacking a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is hydrogen or —OH; R³ is a reversible terminator moiety; R⁴ is adetectable moiety; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof; and L¹⁰⁰ isa divalent linker.

Embodiment 41. The method of any one of embodiments 21 to 40, whereinthe labeled nucleotide comprising a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is hydrogen or —OH; R⁴ is a detectable moiety; B is a divalent cytosineor a derivative thereof, divalent guanine or a derivative thereof,divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof; and L¹⁰⁰ is a divalent linker.

Embodiment 42. The method of embodiment 40 or 41, wherein L¹⁰⁰ is acleavable linker.

Embodiment 43. The method of any one of embodiments 40 to 42, wherein R¹is a triphosphate moiety.

Embodiment 44. The method of any one of embodiments 40 to 43, wherein R²is hydrogen.

Embodiment 45. The method of any one of embodiments 39 to 44, whereinthe reversible terminator comprises an azido moiety, a disulfide moiety,or an alkoxyalkyl moiety.

Embodiment 46. A composition comprising: (a) labeled nucleotidescomprising a free 3′-OH, (b) labeled nucleotides lacking a free 3′-OH,and (c) one or more depleting reagents for decreasing the amount of thenucleotides comprising a free 3′-OH, wherein the one or more depletingreagents comprise: (i) one or more depletion polynucleotides and adepletion polymerase that is active to selectively incorporating thenucleotides comprising a free 3′-OH, wherein the depletionpolynucleotide is free in solution; or (ii) one or more nucleotidecyclases active to selectively cyclize the nucleotides comprising a free3′-OH.

Embodiment 47. The composition of embodiment 46, wherein the compositionis stored in a single container.

Embodiment 48. The composition of embodiment 46 or 47, wherein thecomposition is stored at about 2° C.-8° C., about 20° C.-30° C., orabout 4° C.-37° C.

Embodiment 49. The composition of embodiment 46 or 47, wherein thecomposition is stored at about 4° C. to about 30° C.

Embodiment 50. The composition of any one of embodiments 46 to 49,wherein the depletion polymerase comprises a Klenow fragment, or mutantthereof.

Embodiment 51. The composition of any one of embodiments 46 to 49,wherein the depletion polymerase is a Klenow fragment or mutant thereof,soluble guanylyl cyclase or mutant thereof, or a terminaldeoxynucleotidyl transferase (TdT).

Embodiment 52. The composition of any one of embodiments 46 to 49,wherein the depletion polymerase is active at a temperature of about 1°C. to about 45° C.

Embodiment 53. The composition of any one of embodiments 46 to 49,wherein the depletion polymerase is active at a temperature of about 4°C. to about 37° C.

Embodiment 54. The composition of any one of embodiments 46 to 49,wherein the depletion polymerase is not active above a temperature ofabout 45° C.

Embodiment 55. The composition of any one of embodiments 46 to 54,wherein the one or more depletion polynucleotides comprise a homopolymersequence.

Embodiment 56. The composition of any one of embodiments 46 to 54,wherein the one or more depletion polynucleotides comprises a singlepolynucleotide comprising a hairpin structure and a 5′ overhang.

Embodiment 57. The composition of any one of embodiments 46 to 53,wherein the one or more depletion polynucleotides comprises depletionprimer annealed to a depletion template.

Embodiment 58. The composition of any one of embodiments 46 to 57,further comprising increasing the temperature to a second temperaturerange and reducing the activity of the depletion enzyme.

Embodiment 59. The composition of any one of embodiments 46 to 57,wherein the nucleotides lacking a free 3′-OH comprise a reversibleterminator moiety.

Embodiment 60. The composition of any one of embodiments 46 to 59,wherein the labeled nucleotide lacking a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is hydrogen or —OH; R³ is a reversible terminator moiety; R⁴ is adetectable moiety; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof; and L¹⁰⁰ isa divalent linker.

Embodiment 61. The composition of any one of embodiments 46 to 60,wherein the labeled nucleotide comprising a free 3′-OH has the formula:

wherein R¹ is a polyphosphate moiety, monophosphate moiety, or —OH; R²is hydrogen or —OH; R⁴ is a detectable moiety; B is a divalent cytosineor a derivative thereof, divalent guanine or a derivative thereof,divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof; and L¹⁰⁰ is a divalent linker.

Embodiment 62. The composition of embodiment 60 or 61, wherein L¹⁰⁰ is acleavable linker.

Embodiment 63. The composition of any one of embodiments 60 to 62,wherein R¹ is a triphosphate moiety.

Embodiment 64. The composition of any one of embodiments 60 to 63,wherein R² is hydrogen.

Embodiment 65. The composition of any one of embodiments 59 to 64,wherein the reversible terminator comprises an azido moiety, a disulfidemoiety, or an alkoxyalkyl moiety.

Embodiment 66. A kit comprising the composition of any one ofembodiments 46 to 65.

Embodiment 67. A microfluidic device for sequencing a targetpolynucleotide, comprising: i) a reaction vessel for receiving acomposition of any one of embodiments 46 to 65; ii) one or morereservoirs comprising the composition of any one of embodiments 46 to65; iii) flow paths from each reservoir to the reaction vessel; and iv)a fluidics controller that controls the flow from the reservoir to thereaction vessel.

Embodiment 68. A method of increasing the shelf life of a compositioncomprising modified nucleotides, the method comprising: (a) storing thecomposition of any one of embodiments 46 to 65 at about 1° C. to about40° C. for one or more minutes; and (b) depleting the labelednucleotides comprising a free 3′-OH during said storing, wherein saiddepleting comprises: (i) incorporating with a depleting polymerase thenucleotides comprising a free 3′-OH into one or more depletionpolynucleotides in solution; or (ii) selectively cyclizing thenucleotides comprising the free 3′-OH using a nucleotide cyclase;wherein depleting the nucleotides comprising a free 3′-OH increases theshelf life of the kit comprising modified nucleotides relative to acontrol.

Embodiment 69. A method of decreasing one or more sequencing errors in aplurality of sequencing cycles, said method comprising: (a) contacting acomposition comprising a plurality of labeled nucleotides comprising afree 3′-OH and a plurality of labeled nucleotides lacking a free 3′-OHwith one or more depleting reagents to generate a refined solution,wherein the one or more depleting reagents comprise: (i) a depletionpolynucleotide and a depletion polymerase that is active to selectivelyincorporating the nucleotides comprising a free 3′-OH, wherein thedepletion polynucleotide is free in solution; or (ii) one or morenucleotide cyclases that is active to selectively cyclize thenucleotides comprising a free 3′-OH; (b) inactivating the depletionpolymerase or the one or more nucleotide cyclases; (c) contacting asequencing primer annealed to a target polynucleotide with the refinedsolution and detecting the label of the incorporated labeled nucleotidelacking a free 3′-OH; and repeating step (c), wherein the sequencingerrors is reduced relative to a control.

1.-72. (canceled)
 73. A method of decreasing one or more sequencingerrors in a plurality of sequencing cycles, said method comprising: (a)contacting a composition comprising a plurality of labeled nucleotidescomprising a free 3′-OH and a plurality of labeled nucleotides lacking afree 3′-OH with one or more depleting reagents to generate a refinedsolution, wherein the one or more depleting reagents comprise: (i) adepletion polynucleotide and a depletion polymerase that is active toselectively incorporating the nucleotides comprising a free 3′-OH,wherein the depletion polynucleotide is free in solution; or (ii) one ormore nucleotide cyclases that is active to selectively cyclize thenucleotides comprising a free 3′-OH; (b) inactivating the depletionpolymerase or the one or more nucleotide cyclases; (c) contacting asequencing primer annealed to a target polynucleotide with the refinedsolution and detecting the label of the incorporated labeled nucleotidelacking a free 3′-OH; and repeating step (c), wherein the sequencingerrors are reduced relative to a control. 74.-75. (canceled)
 76. Themethod of claim 73, wherein inactivating the depletion polymerasecomprises heat inactivation or chemical inactivation.
 77. The method ofclaim 73, wherein the depletion polymerase is active at a temperature ofabout 1° C. to about 45° C., or about 4° C. to about 37° C.
 78. Themethod of claim 73, wherein the depletion polymerase comprises a Klenowfragment or mutant thereof.
 79. The method of claim 73, wherein thedepletion polymerase is a Klenow fragment or mutant thereof, solubleguanylyl cyclase or mutant thereof, or a terminal deoxynucleotidyltransferase (TdT).
 80. The method of claim 73, wherein the depletionpolymerase is not active above a temperature of about 45° C.
 81. Themethod of claim 73, wherein the depletion polynucleotide comprises ahomopolymer sequence, or a single polynucleotide comprising a hairpinstructure and a 5′ overhang.
 82. The method of claim 73, whereingenerating the refined solution occurs at a first temperature range ofabout 1° C. to about 45° C.
 83. The method of claim 82, furthercomprising increasing the temperature to a second temperature range andreducing the activity of the depletion polymerase.
 84. The method ofclaim 73, wherein the plurality of labeled nucleotides lacking a free3′-OH comprise a reversible terminator moiety.
 85. The method of claim84, wherein the reversible terminator comprises an azido moiety, adisulfide moiety, or an alkoxyalkyl moiety.
 86. The method of claim 73,wherein the labeled nucleotide lacking a free 3′-OH has the formula:

 wherein R¹ is a triphosphate moiety; R² is hydrogen or —OH; R³ is areversible terminator moiety; R⁴ is a detectable moiety; B is a divalentcytosine or a derivative thereof, divalent guanine or a derivativethereof, divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof, and L¹⁰⁰ is a divalent linker.
 87. The method ofclaim 73, wherein the labeled nucleotide comprising a free 3′-OH has theformula:

 wherein R¹ is a triphosphate moiety; R² is hydrogen or —OH; R⁴ is adetectable moiety; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof, and L¹⁰⁰ isa divalent linker.
 88. The method of claim 86 or 87, wherein L¹⁰⁰ is acleavable linker.
 89. The method of claim 73, wherein the depletionpolynucleotide comprises 2 to 30 consecutive identical nucleotides. 90.The method of claim 73, wherein the depletion polynucleotide comprises adepletion primer hybridized to a depletion template.
 91. The method ofclaim 73, wherein the depleting reagent comprises at least two depletionpolynucleotides, wherein said first depletion polynucleotide comprises ahomopolymer sequence of poly(dA) nucleotides; and said second depletionpolynucleotide comprises a homopolymer sequence of poly(dC) nucleotides.92. The method of claim 91, further comprising a third and a fourthdepletion polynucleotides, wherein said third depletion polynucleotidecomprises a homopolymer sequence of poly(dT) nucleotides; and saidfourth depletion polynucleotide comprise a homopolymer sequence ofpoly(dG) nucleotides.
 93. The method of claim 73, wherein the depletionpolymerase comprises an amino acid sequence that is at least 80%identical to a continuous 500 amino acid sequence within SEQ ID NO: 6.94. The method of claim 73, wherein the labeled nucleotides comprising afree 3′-OH and the labeled nucleotides lacking a free 3′-OH eachcomprise: a first plurality of labeled adenosine triphosphates; a secondplurality of labeled thymidine triphosphates; a third plurality oflabeled guanosine triphosphate; and a fourth plurality of labeledcytosine triphosphates.